Leptin mediates the increase in blood pressure associated with obesity - PubMed (original) (raw)

Graphical abstract

Figure 1

Temporal Relationship between Changes in Weight Gain, Plasma Leptin Concentrations, and Blood Pressure (A) Body weight (g), of mice from 4 weeks of age ad libitum fed either a high-calorie diet (DIO mice) or chow diet (lean mice) for 20 weeks. Following this 20 week period, DIO mice were switched to chow diet for 4 weeks; n = 3–7. (B–E) (B) Plasma leptin concentration (ng/ml), (C) heart rate BPM (beats per min), (D) SBP, and (E) DBP (mmHg) at 2 week intervals, beginning at baseline (4-week-old mice). Following this baseline period, mice were fed a high-calorie diet (DIO) or chow diet (lean) for 20 weeks ad libitum. After 20 weeks of HFD in DIO mice, these mice were switched to chow diet for 4 weeks. n = 3–18. Values mean ± SEM; two-way AVOVA, Bonferroni post hoc test. ∗p < 0.05, ∗∗p < 0.01, and ∗∗p < 0.001.

Figure 2

Leptin Signaling Is Required for the Obesity-Induced Increases in BP (A) Body weight (g) of mice fed 20 weeks a high-calorie (DIO, ob/ob, and db/db mice) or chow (lean ob/ob and db/db mice) diet ad libitum, ∗∗∗p < 0.001 versus lean, +++p < 0.001 versus ob/ob HFD fed, ###p < 0.001 versus db/db HFD (lean, n = 14; DIO, n = 37; ob/ob, n = 8; db/db, n = 3; ob/ob, HFD = 3; db/db, HFD = 5). (B) Average heart rate (BPM) over a 24 hr period of mice fed a high-calorie (DIO mice) or chow (lean mice) diet, ob/ob mice and db/db mice fed a chow diet for over 20 weeks (lean, n = 14; DIO, n = 37; ob/ob, n = 8; db/db, n = 3; ob/ob, HFD = 3; db/db, HFD = 5). (C) Heart rate (BPM) over a 24 hr period in mice fed a chow diet (lean mice), HFD-fed DIO mice, chow-fed ob/ob and db/db mice, and HFD-fed ob/ob and db/db mice. Measurements at 20 weeks are presented (lean, n = 14; DIO, n = 37; ob/ob, n = 8; db/db, n = 3; ob/ob, HFD = 3; db/db, HFD = 5). (D) Average SBP (mmHg) over a 24 hr period of mice fed a high-calorie (DIO mice) or chow (lean mice) diet, ob/ob mice and db/db mice fed a chow diet for 20 weeks (lean, n = 14; DIO, n = 37; ob/ob, n = 8; db/db, n = 3; ob/ob, HFD = 3; db/db, HFD = 5). (E) SBP (mmHg) over a 24 hr period in mice fed a chow diet (lean mice), HFD-fed DIO mice, chow-fed ob/ob and db/db mice, and HFD-fed ob/ob and db/db mice for 20 weeks (lean, n = 14; DIO, n = 37; ob/ob, n = 8; db/db, n = 3; ob/ob, HFD = 3; db/db, HFD = 5). (F) Average DBP (mmHg) over a 24 hr period of mice fed a high-calorie (DIO mice) or chow (lean mice) diet, ob/ob mice and db/db mice fed a chow diet for 20 weeks (lean, n = 14; DIO, n = 37; ob/ob, n = 8; db/db, n = 3; ob/ob, HFD = 3; db/db, HFD = 5). (G) DBP (mmHg) over a 24 hr period in mice fed a chow diet (lean mice), HFD-fed DIO mice, chow-fed ob/ob and db/db mice, and HFD-fed ob/ob and db/db mice (lean, n = 14; DIO, n = 37; ob/ob, n = 8; db/db, n = 3; ob/ob, HFD = 3; db/db, HFD = 5). (H) Plasma leptin (ng/ml) correlates with heart rate (BPM). Linear regression, r2 = 0.5855; p < 0.001; n = 24. (I) Plasma leptin (ng/ml) correlates with SBP (mmHg). Linear regression, r2 = 0.6205; p < 0.001; n = 24. (J) Plasma leptin (ng/ml) correlates with DBP. Linear regression, r2 = 0.5883; p < 0.001; n = 24. (K) Exogenous leptin administration into ob/ob mice increased heart rate. Vehicle (Veh) treatment into ob/ob mice and leptin administration into db/db mice produced no change. Two-way ANOVA, Bonferroni post hoc test. n = 3–8. (L) Average heart rate (BPM) over 11 day treatment period of leptin-treated ob/ob mice, compared to the baseline HR of ob/ob mice, the baseline of DIO and lean mice. One-way ANOVA, Bonferroni post hoc test, paired t test between leptin-treated ob/ob mice and ob/ob baseline, n = 8–37. (M) Exogenous leptin administration into ob/ob mice, increased SBP (mmHg). Vehicle treatment into ob/ob mice and leptin administration into db/db mice produced no change. Values represent mean ± SEM. Two-way ANOVA, Bonferroni post hoc test. n = 3–8. (N) Average SBP (mmHg) over 11 day treatment period of leptin-treated ob/ob mice compared to ob/ob baseline SBP, DIO, and lean baseline SBP measurements. One-way ANOVA, Bonferroni post hoc test, paired t test between leptin-treated ob/ob mice and ob/ob baseline. n = 8–37. (O) Exogenous leptin administration into ob/ob mice increased diastolic BP (DBP) (mmHg). Vehicle treatment into ob/ob mice and leptin administration into db/db mice produced no change. Two-way ANOVA, Bonferroni post hoc test. n = 3–8. (P) Average DBP (mmHg) over 11 day treatment period of leptin-treated ob/ob mice compared to ob/ob baseline DBP, DIO baseline DBP, and lean mice DBP. One-way ANOVA, paired t test between leptin-treated ob/ob mice and ob/ob baseline. n = 8–37. Values represent mean ± SEM, one-way AVOVA, Bonferroni post hoc test, unless specifically stated. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. See also Figure S1.

Figure S1

Characterization of Leptin Effects on Metabolic and Cardiovascular Parameters, Related to Figure 2 (A) The average 12h dark and 12h light heart rate (HR) of lean, DIO, chow fed ob/ob, db/db and HFD fed ob/ob, db/db mice. Mean ± SEM, Two way ANOVA, ∗p < 0.05, ∗∗∗p < 0.001 Vs DIO, (lean n = 14, DIO n = 37, ob/ob n = 8, db/db n = 3, ob/ob HFD = 3, db/db HFD = 5) (B) The average 12h dark and 12h light systolic BP (SBP) of lean, DIO, chow fed ob/ob, db/db and HFD fed ob/ob, db/db mice. Mean ± SEM, Two way ANOVA, ∗p < 0.05, ∗∗p < 0.01 ∗∗∗p < 0.001 Vs DIO, (lean n = 14, DIO n = 37, ob/ob n = 8, db/db n = 3, ob/ob HFD = 3, db/db HFD = 5) (C) The average 12h dark and 12h light diastolic BP (DBP) of lean, DIO, chow fed ob/ob, db/db and HFD fed ob/ob, db/db mice. Mean ± SEM, Two way ANOVA, ∗p < 0.05, ∗∗p < 0.01 ∗∗∗p < 0.001 Vs DIO, (lean n = 14, DIO n = 37, ob/ob n = 8, db/db n = 3, ob/ob HFD = 3, db/db HFD = 5) (D) Average activity (arbitrary units) throughout 24hr period of mice fed a high calorie (DIO mice) or chow (lean mice) diet, ob/ob mice and db/db mice fed a chow diet for 20 weeks. Values mean ± SEM, (lean n = 14, DIO n = 37, ob/ob n = 8, db/db n = 3, ob/ob HFD = 3, db/db HFD = 5) (E) Average Activity (arbitrary units) over a 24hr period in mice fed a chow diet (lean mice), HFD fed DIO mice, chow fed ob/ob and db/db mice, HFD fed ob/ob and db/db mice. Values mean ± SEM, One-way AVOVA, Bonferroni post hoc test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, (lean n = 14, DIO n = 37, ob/ob n = 8, db/db n = 3, ob/ob HFD = 3, db/db HFD = 5) (F) Plasma leptin concentration of lean mice baseline, DIO mice baseline, db/db mice baseline, ob/ob mice baseline and the plasma leptin concentration after 11 days of treatment in ob/ob mice. Mean ± SEM, one way ANOVA, ∗∗p < 0.01, ∗∗∗p < 0.001, n = 3-12 (G) Plasma insulin (ng/ml) does not correlate with heart rate (BPM) in mice on a C57Bl/6J background. Linear regression, r2 = 0.0004055, p = 0.9368, n = 18 (H) Plasma insulin (ng/ml) does not correlate with systolic BP (SBP) in mice on a C57Bl/6J background. Linear regression, r2 = 0.0003732, p = 0.9394, n = 18 (I) Plasma insulin (ng/ml) does not correlate with diastolic BP (DBP) in mice on a C57Bl/6J background. Linear regression, r2 = 0.003801, p = 0.8080, n = 18 (J) 24hr circadian heart rate (BPM) rhythm in leptin treated ob/ob mice, baseline DIO mice measurements and ob/ob baseline mice measurements, day 4 of treatment. Values mean ± SEM n = 8-37 (K) 24 hr circadian systolic BP (SBP) (mmHg) rhythm in leptin treated ob/ob mice, the baselines of DIO and ob/ob mice, day 4 of treatment. Values mean ± SEM, n = 8-37 (L) 24 hr circadian diastolic BP (DBP) (mmHg) rhythm in leptin treated ob/ob mice, the baselines of DIO and ob/ob mice day 4 of treatment. Values mean ± SEM, n = 8-37 (M) Leptin administration to ob/ob mice decreases BW, while vehicle administration into ob/ob mice and leptin administration into db/db mice produced no change in bodyweight. Values mean ± SEM. Two-way ANOVA, ∗∗∗p < 0.001, n = 3-8 (N) Change in food intake in exogenous leptin treated ob/ob mice, vehicle treated ob/ob mice and leptin treated db/db mice. Values mean ± SEM. Two-way ANOVA. (Significance between ob/ob leptin treatment and ob/ob vehicle treated, day 2 p < 0.05, day 4p < 0.05, day 5 p < 0.001, day 11 p < 0.05). (Significance between ob/ob leptin treatment and db/db leptin treated, day 2 p < 0.01, day3 p < 0.05, day 5 p < 0.05, day 6 p < 0.01, day 7p < 0.05, day 8 p < 0.05, day 9 p < 0.01, day 10 p < 0.05, day 11 p < 0.01), n = 3-8 (O) Average activity (arbitrary units) over 11 day treatment period in exogenously leptin administrated ob/ob mice, compared with the baseline activity of ob/ob, DIO and lean mice. Values mean ± SEM. One way ANOVA, ∗p < 0.05, ∗∗∗p < 0.001 n = 8-37. Additional data for Figure 2.

Figure S2

Effects of Leptin Receptor Antagonist on Cardiovascular and Immunohistochemical Parameters, Related to Figure 3 (A) 24 hr heart rate (HR) (BPM) circadian rhythm in DIO mice on day 7 treated in the lateral ventricle (LV) with leptin receptor antagonist (LRA) treatment, compared to the baseline of DIO and lean mice. Mean ± SEM, n = 6-47 (B) 24 hr circadian systolic BP (SBP) (mmHg) in DIO mice on day 7 from treatment in the lateral ventricle (LV) with leptin receptor antagonist (LRA) compared to the baseline of DIO and lean mice. Mean ± SEM, n = 6-37 (C) 24 hr circadian rhythm of diastolic BP (DBP) (mmHg) in DIO mice on day 7 of treatment in the lateral ventricle (LV) with leptin receptor antagonist (LRA) compared to the baselines of DIO and lean mice at baseline. Mean ± SEM, n = 6-37 (D) pSTAT3 expression in the ARH region of DIO mice, following either 0.5 μl of aCSF or leptin antagonist (5μg/μl) ICV injection and then either peripheral administration of Vehicle or leptin (2μg/g). One way ANOVA, Bonferroni post hoc, n = 3-5 (E) pSTAT3 expression in the VMH of DIO mice, following either 0.5 μl of aCSF or leptin antagonist (5μg/μl) ICV injection and then either peripheral administration of Vehicle or leptin (2μg/g). One way ANOVA, Bonferroni post hoc test. ∗p < 0.05, n = 3-5 (F) pSTAT3 Immunochemistry representative figure of hypothalamic sections of DIO mice, following either 0.5 μ of aCSF or leptin antagonist (5uμg/μl) ICV injection and then either peripheral administration of Vehicle or leptin (2μg/g). The DMH analysis can be found in Lee et al. (2013). Additional data for Figure 3.

Figure 3

Blockade of Leptin Actions in DIO Mice Reduces Elevated BP and HR (A–C) (A) Percentage change in HR, (B) SBP, and (C) DBP over a 5 day treatment period with either leptin antibody or vehicle (IgG control). Student’s t test, n = 4–8. (D) Percentage daily change in body weight of DIO mice before and during 5 days of treatment and posttreatment of leptin antibody or vehicle (IgG control). Two-way ANOVA, Bonferroni post hoc test. n = 4–8. (E) Daily change in HR (BPM) in DIO mice treated LV with aCSF or leptin receptor antagonist (LRA). Two way ANOVA, Bonferroni post hoc, n = 4-6 (F) Total 24hr HR(BPM) after 7 days of LRA treatment in DIO mice compared to lean and DIO mice baselines. t test paired (DIO baseline versus LRA), unpaired t test. n = 6–37. (G) Daily change in SBP (mmHg) from baseline in DIO mice treated in the LV with aCSF or LRA. Two-way ANOVA, Bonferroni post hoc test, n = 4–6. (H) Total 24 hr SBP (mmHg) after 7 days of LRA treatment in DIO mice compared to the baselines of lean and DIO mice. t test paired (DIO baseline versus LRA), unpaired t test. n = 6–37. (I) Daily change in DBP (mmHg) in DIO mice treated with LVaCSF or LRA. Two-way ANOVA, Bonferroni post hoc, n = 4–6. (J) Total 24 hr DBP (mmHg) after 7 days of LRA in DIO mice compared to the baseline measurements of lean and DIO mice. t test paired (DIO baseline versus LRA), unpaired t test. n = 6–37. (K) Average food intake over the 7 day treatment period of LVaCSF (control) or LRA DIO mice. t test, n = 4–6. Values represent mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. See also Figure S2.

Figure 4

The DMH Is Involved in Leptin-Mediated Increases in HR and BP (A) Daily change in HR (BPM) from baseline in DIO mice treated in the DMH with aCSF or a LRA. Two-way ANOVA, Bonferroni post hoc test, n = 4. (B) Total 24 hr HR (BPM) after 7 days of LRA treatment in the DMH of DIO mice compared to lean and DIO mice at baseline. t test paired (DIO baseline versus LRA), unpaired t test. n = 4–37. (C) Daily change in SBP (mmHg) from baseline in DIO mice treated in the DMH with aCSF or a LRA. Two-way ANOVA, Bonferroni post hoc test, n = 4. (D) Total 24 hr SBP (mmHg) after 7 days of LRA treatment in the DMH of DIO mice compared to lean and DIO mice at baseline. t test paired (DIO baseline versus LRA), unpaired t test. n = 4–37. (E) Daily change in DBP (mmHg) from baseline in DIO mice treated in the DMH with aCSF or a LRA. Two-way ANOVA, Bonferroni post hoc test, n = 4. (F) 24 hr DBP (mmHg) after 7 days of LRA treatment in the DMH of DIO mice compared to lean and DIO mice at baseline. t test paired (DIO baseline versus LRA), unpaired t test. n = 4–37. (G) Percentage change in SBP (mmHg) of DIO mice, following control (scrambled AAV) or leptin receptor knockdown AAV into the DMH. Two-way ANOVA, Bonferroni post hoc test, n = 4–6. (H) Quantitative analysis from day 31 onward of AAV scrambled control-injected DIO mice and AAV LepR knockdown-treated DIO mice. t test, n = 4–6. (I) 24 hr circadian rhythm of SBP (mmHg) at baseline and then following the seventh day of AAV Cre injection into the DMH of LepR flox mice. Two-way ANOVA, Bonferroni post hoc test, n = 4. (J) 24 hr circadian rhythm of heart rate (BPM) at baseline and then following the seventh day of AAV Cre injection into the DMH of LepR flox TB mice. Two-way ANOVA, Bonferroni post hoc test, n = 4. (K) 24 hr circadian rhythm of SBP (mmHg) at baseline and then following the seventh day of AAV Cre injection into the DMH of LepR flox TB mice. Two-way ANOVA, Bonferroni post hoc test, n = 4. Values represent mean ± SEM. ∗p < 0.05 and ∗∗p < 0.01. See also Figure S3.

Figure S3

Cardiovascular Effects of Altering Leptin Signaling in the DMH, Related to Figure 4 (A) 24hr heart rate (BPM) circadian rhythm in DIO mice on day 7 of leptin receptor antagonist (LRA) treatment into the DMH, compared to the baseline of DIO and lean mice. Mean ± SEM, n = 4-37 (B) 24hr systolic BP (SBP) (mmHg) circadian rhythm in DIO mice on day 7 of leptin receptor antagonist (LRA) treatment into the DMH, compared to the baseline of DIO and lean mice. Mean ± SEM, n = 4-37 (C) 24hr diastolic BP (DBP) (mmHg) circadian rhythm in DIO mice on day 7 of leptin receptor antagonist (LRA) treatment into the DMH, compared to the baseline of DIO and lean mice. Mean ± SEM, n = 4-37 (D) Representative image of GFP fluorescence, indicating AAV infection, to knockdown leptin receptor in the DMH of DIO mice. Virus had been injected 55 days prior to sacrifice of mice. Additional data for Figure 4.

Figure 5

Leptin Excites Leptin-Receptor-Expressing Neurons in the DMH (A) Whole-cell patch-clamp electrophysiology in LepR-Cre-YFP mice; bath application of leptin (100 nM) induced an increase in spontaneous firing rate. (B) Voltage-current relations from the cell shown in Figure S4A before and following exposure to leptin (100 nM). Note the transient rebound depolarization at the offset of the response to hyperpolarizing current injection and its prolongation in the presence of leptin, illustrated below with responses to hyperpolarizing current injection in control (black) and in the presence of leptin (red). Right, plot of data shown in (B); note the decreased slope of the voltage-current relations in the presence of leptin (100 nM) (open triangles) compared to control (closed squares), indicating a decrease in neuronal input resistance and extrapolated reversal potential around −35 mV, consistent activation of a nonselective cation conductance. (C) Averaged 5 min (data collected every 10 s) heart rate (BPM) of DIO mice injected in the DMH with inhibitor chimeric ion channel virus (pSyn::Flex-rev-PSAM L141F:GlyR-IRES-GFP) and treated i.p. with vehicle or PSEM (5 mg/kg). n = 5. (D) Average (20–180 min) heart rate (BPM) over treatment period of DIO mice injected in the DMH with inhibitor chimeric ion channel virus with vehicle or PSEM. Paired t test, n = 5. (E) Averaged 5 min (data collected every 10 s) SBP (mmHg) of DIO mice injected in the DMH with inhibitor chimeric ion channel virus and treated i.p. with vehicle or PSEM (5 mg/kg). Two-way ANOVA, Bonferroni post hoc test, n = 5. (F) Average (20–180 min) SBP (mmHg) in DIO mice injected in the DMH with inhibitor chimeric ion channel virus in mice treated i.p. with Veh or pharmacologically selective effector molecule (PSEM). Paired t test, n = 5. (G) Averaged 5 min (data collected every 10 s) DBP (mmHg) of DIO mice injected in the DMH with inhibitor chimeric ion channel virus and treated i.p. with vehicle or PSEM (5 mg/kg). Two-way ANOVA, Bonferroni post hoc test, n = 5. (H) Average (20–180 min) DBP (mmHg) of DIO mice treated with chimeric ion channel virus and with Veh or PSEM. Paired t test, n = 5. (I) Average daily HR (BPM) in DIO mice injected in the DMH with inhibitor chimeric ion channel virus treated i.p. with Veh or PSEM for 3 days. Paired t test, n = 5. (J) Average daily SBP (mmHg) in DIO mice treated with inhibitor chimeric ion channel virus and with Veh or PSEM for 3 days. Paired t test, n = 5. Values represent mean ± SEM. ∗p < 0.05. See also Figure S4.

Figure S4

Leptin Activates DMH Neurons to Regulate Blood Pressure, Related to Figure 5 (A) In DMH lepR expressing neurons, leptin (100nM) induced membrane depolarization, increased firing rate and underlying oscillations in membrane potential to close to threshold. Oscillations induced in the presence of leptin shown on an expanded time-scale below (2) compared with control (1). Note oscillations were reset and abolished following membrane hyperpolarization with constant current injection (3). (B) Leptin (100nM) induced excitation characterized by an increase in firing frequency and spontaneous excitatory postsynaptic potentials (EPSPs), the latter illustrated on an expanded scale below and superimposed. (C) Average daily diastolic BP (DBP) (mmHg) in DIO mice treated with chimeric ion channel virus (pSyn::Flex-rev-PSAM L141F:GlyR-IRES-GFP) and with Veh or PSEM, for 3 days. Mean ± SEM, Paired t test, ∗∗p < 0.01 n = 5 (D) Average daily heart rate (HR) (BPM) in lean mice treated with activator chimeric ion channel virus (pSyn::Flex-rev-PSAM Y115F:5HT3 HC-IRES-GFP) in the DMH and treated with Veh or PSEM, for 3 days. Mean ± SEM, two way ANOVA, ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05, n = 4 (E) Average daily systolic BP (SBP) (mmHg) in lean mice treated with activator chimeric ion channel virus (pSyn::Flex-rev-PSAM Y115F:5HT3 HC-IRES-GFP) in the DMH and treated with Veh or PSEM, for 3 days. Mean ± SEM, two way ANOVA, n = 4 (F) Average daily diastolic BP (DBP) (mmHg) in lean mice treated with activator chimeric ion channel virus (pSyn::Flex-rev-PSAM Y115F:5HT3 HC-IRES-GFP) in the DMH and treated with Veh or PSEM, for 3 days. Mean ± SEM, two way ANOVA, ∗p < 0.05, n = 4 (G) Average daily HR (BPM) in DIO mice before injection to inhibitor in the DMH and treated i.p. with Veh or PSEM. Mean ± SEM, two way ANOVA, n = 3 (H) Average daily SBP (mmHg) in DIO mice before injection of inhibitor in the DMH and treated with i.p. Veh or PSEM. Mean ± SEM, two way ANOVA, n = 3. Additional data for Figure 5.

Figure 6

Blood Pressure Measurements in Humans with Leptin and Leptin Receptor Mutations (A) Leptin-deficient children (n = 8) have significantly lower SBP (mmHg) compared to age- and weight-matched control subjects (n = 42). (B) Leptin-receptor-deficient children (n = 12) have significantly lower SBP (mmHg) compared to age- and weight-matched control subjects (n = 48). (C) Baseline pretreatment mean arterial pressure (MAP) (mmHg) of leptin-deficient adult and control subjects. n = 3. (D) Change in MAP (mmHg) after 19 weeks of weight loss treatment. n = 3. Value represent mean ± SEM. Student’s t test. ∗p < 0.05. See also Figure S5 and Table S1.

Figure S5

Heart Rate Response to Leptin or Lifestyle-Induced Weight Loss, Related to Figure 6 Change in heart rate (HR) (BPM) after 19 weeks of weight loss in leptin deficient adults treated with recombinant human leptin and BMI matched controls with the same degree of weight loss. Mean ± SEM, n = 3. Additional data for Figure 6.