Partial Leptin Reduction as an Insulin Sensitization and Weight Loss Strategy

Abstract

The physiological role of leptin is thought to be a driving force to reduce food intake and increase energy expenditure. However, leptin therapies in the clinic have failed to effectively treat obesity, predominantly due to a phenomenon referred to as leptin resistance. The mechanisms linking obesity and the associated leptin resistance remain largely unclear. With various mouse models and a leptin neutralizing antibody, we demonstrated that hyperleptinemia is a driving force for metabolic disorders. A partial reduction of plasma leptin levels in the context of obesity restores hypothalamic leptin sensitivity and effectively reduces weight gain and enhances insulin sensitivity. These results highlight that a partial reduction in plasma leptin levels leads to improved leptin sensitivity, while pointing to a new avenue for therapeutic interventions in the treatment of obesity and its associated comorbidities.

Keywords: diabetes; hypothalamus; leptin; leptin resistance; obesity.

Figure 1:. Increasing leptin levels in obese mice exacerbates obesity and metabolic dysfunction.

Leptin gene expression in various fat depots collected from wildtype (WT) mice (n = 5 per time point) transferred from a chow diet to a HFD (A). Circulating leptin levels in WT mice (n = 10) from chow diet to HFD (B); ALep-TG (n = 10) and littermate control (n = 11) mice at 8-weeks of age were placed on HFD for 6 weeks and then switched to HFD diet with Dox (600mg/kg). Leptin (C), insulin (D) and adiponectin (E) levels were measured before and after supplementing DOX in the diet, and leptin levels were normalized to total fat mass. Body weight gain (F), fat mass (G), lean mass (H), oral glucose tolerance tests (OGTTs) before (I) and after (J) DOX diet as well as insulin tolerance tests (ITTs) (K) after DOX were done in ALep-TG and littermate Ctrl mice. Histological analysis with a H&E stain of livers (L) and brown fat (M) were assessed. (Data are given as mean ± SEM. Error bars indicate SEM. *p < 0.05; **p < 0.01; ***p < 0.001).

Figure 2:. Partial leptin reduction in mice protects against diet-induced obesity.

Cas9-sgLeptin and littermate control mice at 8 weeks of age were placed on HFD with Dox 600 for 10 weeks. (A) Circulating leptin levels per total fat mass in Cas9-sgLeptin (n = 5) and littermate control mice (n = 5) at the indicated time points; (B) body weight gain during HFD feeding in Cas9-sgLeptin (n = 10) and littermate control mice (n = 10). (C) OGTTs on Cas9-sgLeptin (n = 10) and control mice (n = 10). Area under the curve (AUC) was calculated and inserted inside this figure; Alepflox-HZ and littermate control mice at 8 weeks of age were placed on HFD with Dox 600 for 9 weeks. Two different cohorts of mice were used in this study. circulating leptin (D) and adiponectin (E) levels measured in the first cohort of Alepflox-HZ (n = 7) and control mice (n = 5); (F) body weight gain in ALepflox-HZ (n = 12) and control mice (n = 10); (G) OGTT in ALepflox-HZ (n = 12) and control mice (n = 10). After euthanizing the mice, brown fat (H) and liver (I) were processed for H&E staining. (Data are given as mean ± SEM. Error bars indicate SEM. *p < 0.05; **p < 0.01; ***p < 0.001).

Figure 3:. Partial, not complete reduction of circulating leptin, protects mice from obesity.

ALepKO and littermate control mice were placed on HFD supplemented with two different amounts of Doxycycline (DOX) (600mg/kg (DOX600) and 10mg/kg (DOX10)). Body weights, circulating leptin levels, OGTTs, ITTs and histological analysis were performed. (A) Body weight gain of ALepKO (n = 6) and littermate controls (n = 7) on DOX600; (B) body weight gain ALepKO (n = 7) and littermate controls (n = 7) on DOX10; (C) Circulating leptin levels per total fat mass of ALepKO (n = 7) and littermate controls (n = 6) before and after DOX 600; (D) Circulating leptin levels per total fat mass of ALepKO (n = 9) and littermate controls (n = 8) on DOX10; (E) OGTT on ALepKO (n = 8) and littermate controls(n = 6) on DOX600; (F) OGTTs on ALepKO (n = 6) and littermate controls (n = 11) on DOX10; (G) ITTs on ALepKO (n = 6) and littermate controls (n = 6) DOX600; (H) ITTs on ALepKO (n = 6) and littermate controls (n = 8) on DOX 10; (I) Brown adipose tissue histology on DOX600; (J) Brown adipose tissue histology on DOX10; (K) Oxygen consumption (VO₂) of ALepKO (n = 6) and littermate controls (n = 6) on DOX10 in metabolic cages; (L) Locomotor activity of ALepKO mice (n = 6) and littermate controls (n = 6) on DOX10 during the dark period, daytime and across the entire 24hr period. (Data are given as mean ± SEM. Error bars indicate SEM. *p < 0.05; **p < 0.01; ***p < 0.001).

Figure 4.. Decreasing leptin levels with neutralizing anti-leptin antibodies reduces body weight gain and liver steatosis.

A cohort of obese mice (n = 8 per group) were treated either with control antibody or leptin neutralizing antibody (hLep3) for two weeks. Antibody injections were done every other day. Body weight (A) and food intake (C) were measured before each injection. Body weight gain was calculated (B); OGTTs were performed before (D) and (E) after antibody injection; Total fat mass (F) was measured by Eco-MRI. For the metabolic cage studies, obese WT mice (n = 6 per group) were treated with a control antibody (hIGG) or hLep3 antibody. (G) Food consumption measured in metabolic cages after vehicle or hLep3 treatment; (H) RER measured in vehicle and hLep3 treated mice; After a two-week treatment period, mice were euthanized and brown fat and liver were collected for histology analysis. H&E staining of brown adipose tissue (I) and liver (J); Obese WT mice (n = 5 per group) were housed in thermoneutral chambers and treated with control antibody (hIGG) or hLep3 neutralizing antibody for two weeks (K) Effects of the neutralizing antibody hLep3 on body weight, body weight gain and OGTTs on mice housed at thermoneutrality; (L) Effect of hLep3 on body weight gain in inducible ob/ob mice. (Data are given as mean ± SEM. Error bars indicate SEM. *p < 0.05; **p < 0.01; ***p < 0.001).

Figure 5.. Leptin sensitivity is inversely correlated with circulating leptin levels.

Expression of pomc (A) and socs3 (B) in the ARH region of control (n = 12) and AlepKO mice (n = 12); (C) Daily food intake was measured in control (n = 7) and ALepKO mice (n = 7) during PBS or a low dose Doxycycline (5mg/kg body weight) oral gavage period; (D) Effects of acute leptin injections on food intake in ALepKO (n = 8) and littermate control mice (n = 9) after overnight fasting; (E) DAB staining of p-STAT3 after leptin injection in ALepKO and Ctrl mice; (F) Gene expression of pomc in ARH region after neutralizing leptin antibody treatment; (G) Gene expression of socs3 in ARH region after neutralizing leptin antibody treatment; (H) DAB staining of p-STAT3 after leptin injection in neutralizing hLEP3 treated mice; (I) Effects on food intake in obese WT mice (vehicle (n = 7) vs mLep3 (n = 6)) were chronically treated with control antibodies (mIGG) or a mouse version of the neutralizing leptin antibody (mLep3); (J-N) Brightfield illumination (J) of a POMC neuron that expresses leptin receptors from POMC-hrGFP::LepR-cre::tdtomato mice; (K) and (L) show the same neuron under FITC (hrGFP, green cell) and Alexa Fluor 594 (tdtomato, red cell) illumination. Complete dialysis of Alexa Fluor 350 from the intracellular pipette is shown in (M) and a merged image of a POMC neuron targeted for electrophysiological recording (N); Merged image. (Arrow indicates the targeted cell. Scale bar = 50 μm); (O) Representative electrophysiological trace demonstrating a Leptin receptor expressing POMC neuron from chow diet-fed mice is depolarized by leptin (100 nM); (P) Representative electrophysiological trace demonstrating a leptin receptor expressing POMC neuron from HFD feeding mice is depolarized by leptin (100 nM); (Q) Representative electrophysiological trace demonstrating a Leptin receptor expressing POMC neuron from HFD feeding mice which is injected with neutralizing antibody is depolarized by leptin (100 nM); (R) Histogram illustrates the acute effects of leptin (100 nM) on the membrane potential of leptin receptor expressing POMC neurons from chow or HFD feeding mice with or without antibody injection. (Data are given as mean ± SEM. Error bars indicate SEM. *p < 0.05; **p < 0.01; ***p < 0.001).

Figure 6.. Summary of relationship of circulating leptin levels and leptin sensitivity.

(A) With adipose tissue expansion, high leptin levels are achieved in circulation, leading to a high degree of leptin resistance; (B) Reducing leptin levels in a setting of high circulating leptin restores leptin sensitivity.