Tirzepatide retention in iWAT with mesoporous polydopamine encapsulation enhances weight loss through leptin receptor signaling

Graphical abstract

Highlights

• •MPDA encapsulation of TZP increases its retention time in iWAT of mice by iWAT injection.
• •MPDA encapsulation of TZP shows more dramatic body weight reduction in DIO mice by iWAT injection.
• •MPDA encapsulation of TZP reduces leptin expression and increases BCAA catabolism in iWAT of DIO mice by iWAT injection.
• •TZP treatment improves leptin receptor signaling in iWAT both in vivo and ex vivo.
• •Leptin receptor mediates is involved in body weight reduction by iWAT injection of MPDA encapsulated TZP.

Abstract

Introduction

Tirzepatide (TZP), a dual glucose-dependent insulinotropic polypeptide/glucagon-like peptide 1 receptor agonist, has been showing superior benefits in weight loss and glucose lowering in patients with obesity, while the tissue-specific mechanisms of TZP as well as its side effects remain to be investigated.

Objectives

We aimed to explore whether localized injection of TZP into inguinal white adipose tissue (iWAT) would be as effective as subcutaneous injection for weight loss, in order to understand the local effects and signaling pathways of TZP for precision medicine.

Methods

Mesoporous polydopamine was synthesized and mixed with TZP for encapsulation (MPDA@TZP), followed by characterization of its retention in iWAT after local injection. Both diet-induced obesity (DIO) mice and db/db mice received local injection of TZP, and transcriptomic analysis of iWATs was performed. iWATs were also dissected for ex vivo assays.

Results

MPDA@TZP successfully increased the retention time of TZP in the iWAT of mice and had a more dramatic effect on weight loss and improvement in plasma lipid profiles compared to TZP alone in DIO mice, while showing comparable glucose-lowering efficacy. Transcriptomic analysis indicated that iWAT injection of MPDA@TZP improved leptin resistance, beiging, lipid metabolism, mitochondrial activity and branched-chain amino acid (BCAA) catabolism in iWAT. Leptin receptor deficiency in db/db mice abolished the weight reduction effect of MPDA@TZP via iWAT local injection compared to that in DIO mice, while the glucose-lowering effects were comparable in both db/db and DIO mice.

Conclusion

These findings indicate that the retention of TZP in iWAT via MPDA encapsulation amplified its effect on weight loss in mice through leptin receptor signaling compared with TZP alone, which provides new insights into the tissue-specific mechanism and alternative delivery strategies of TZP for targeting specific WAT tissues.

Introduction

Over the past decades, the prevalence of obesity worldwide has significantly increased, while the therapeutics remained limited [1]. Obesity comes with increasing health issues, such as type 2 diabetes mellitus (T2DM), chronic renal failure, non-alcoholic fatty liver disease, musculoskeletal disorders, cardiovascular dysfunction, etc. [2]. Lifestyle intervention (physical exercise, low-calorie diet, consultation) is central to weight loss, yet only ∼ 40 % of individuals achieve ≥ 5 % weight loss [3,4]. Pharmacological therapy is also recommended for patients with BMI 27––30 kg/m² or ≥ 30 kg/m² BMI with more than one obesity-related complication [5]. Currently, approved anti-obesity drugs induce ∼ 5 % weight loss over 6–12 months, with medication restricted based on potential side effects [6]. For patients with severe obesity-related diseases, metabolic surgery is the most efficient intervention to improve serum lipids, long-term survival, as well as other outcomes [7,8].

In the last few decades, glucagon-like peptide-1 receptor agonists (GLP-1RAs) have emerged as the most effective medications for patients with T2DM and/or obesity [9]. Despite of the improvements with GLP-1RAs treatment, many patients still do not achieve the ideal therapeutic goals due to the poor long-time compliances, gastrointestinal side effects, increased thyroid cancer and pancreatitis risks, as well as heterogeneous patient responsiveness [10]. Therefore, it remains necessary to develop agents that could enhance or complement the therapeutic effects of GLP-1RA efficacy. White and brown adipose tissues (WAT and BAT) are key sites of energy storage and expenditure, respectively, and their dysfunction is associated with the pathogenesis of obesity and T2DM [11]. Therefore, one of the strategies to enhance the clinical effect of GLP-1RAs is to strengthen the potential local effect of GLP-1RAs in WAT or BAT. Glucose-dependent insulinotropic polypeptide (GIP), a hormone released from the gastrointestinal tract after food consumption, acts on GIP receptors (GIPRs) that are highly expressed in WAT and BAT [[12], [13], [14]]. Similarly, long-acting GIP analogues could also improve both insulin secretion and weight loss [[15], [16], [17]].

Given the vast global burden of obesity and its complications, dual- or triple-agonists that simultaneously engage GIP and GLP-1 receptors represent the most promising next-generation therapeutics [16]. Tirzepatide (TZP) is a dual GLP-1R/GIPR agonists and has excellent effects on weight loss and glycemic control in individuals with T2DM or obesity compared with the monotherapy of GLP-1R or GIPR agonists [18,19]. Interestingly, pair-feeding analysis of mice with diet induced obesity (DIO) suggests that TZP treatment could provide weight-dependent and −independent improvement in glycemic control [16]. Besides, the benefit of TZP on glucose regulation in patients with T2DM is affected by weight-independent insulin sensitivity [20]. In insulin-resistant mice with obesity, TZP improves insulin sensitivity to a better degree than GLP-1RA alone with independent of body weight loss [21]. Clinically, TZP could improve fasting insulin and glucose levels, and induce dramatic and persistent weight loss of approximately 20 % in patients with obesity; however, only 56.7 % of the patients achieved the desired effects [22]. Besides, patient compliance is challenging due to the side effects, easy rebound, financial burden or shortage in supply [23]. However, these clinical findings have inspired interest in exploring the mechanisms through which TZP achieves its therapeutic effects, as well as the delivery strategies that would benefit more patients and reduce the potential side effects through precise medicine.

Normally, TZP is administered via subcutaneous (SC) injection into the loose connective tissue beneath the skin (including minimal fat) and then it enters systemic circulation through subcutaneous capillaries. It has been reported clinically that TZP’s effect on body weight loss might be primarily related to its effect on reducing energy intake and appetite [24]. Besides, it has also been reported that GIPR activation on adipocytes contributed to weight loss in mouse models [25]. Thus, we proposed that local treatment in WAT with TZP might play beneficial roles in metabolic profiles in mice while reducing the potential side effects in the central nervous system or intestine. The development of WAT local delivery technology has provided opportunities to develop biological agents for the treatment of obesity, which could increase local concentration, reduce side effects and lower drug dosages [26]. Nanoparticles have the characteristics of surface functionalization, biocompatibility, superior degradability, and targeting abilities, and are widely used in the fields of drug delivery, tissue repair and anti-tumor therapy. Mesoporous polydopamine (MPDA) nanoparticles, have shown excellent performance in carrier properties, strong adhesive abilities, preparation simplicity, and biocompatibility. The mesoporous structure of MPDA provides a high capacity for molecule loading and contains multiple functional groups, including amine, catechol, and imine, for chemical modification [27]. Previous reports have demonstrated that the encapsulation of drugs like doxorubicin in MPDA particles effectively kills cancer cells with high payload capacity [28]. However, the application of MPDA nanoparticles in metabolic diseases remains largely underexplored. This study aimed to enhance the metabolic benefits of TZP through MPDA encapsulation via iWAT local injection, which would enable sustained retention, prolonged local release, and extended action time of TZP in iWAT.

In this study, using high-fat diet (HFD)-fed and db/db mouse models, we reveal that iWAT delivery of TZP by MPDA encapsulation leads to significant weight loss, which is associated with enhanced lipid catabolism, increased mitochondrial metabolism, and elevated branched chain amino acids (BCAAs) catabolism via LepR signaling in iWAT. Our findings provide new perspectives on the functions of TZP and other polypeptides with the aid of nanomaterials, which will guide their clinical applications in precision medicine in future.

Materials and methods

Preparation of MPDA nanoparticles

For preparing MPDA nanoparticles, 0.1 g block copolymer F127 (CAS: 9003–11-6, Sigma-Aldrich) and 0.15 g dopamine hydrochloride (CAS:62–31-7, Aladdin Industrial Inc) were added into a stirring mixed buffer containing ethanol (5 mL) (CAS:64–17-5, Macklin) and deionized water (5 mL). Next, TMB (0.16 mL) (CAS: 108–67-8, Macklin) was added to the mixture above and sonicated for 6 mins with a water bath. Subsequently, ammonia (0.375 mL) (CAS:1336–21-6, Macklin) was also dispersed in the mixed buffer with stirring. In the end, MPDA nanoparticles were obtained with centrifugation at 9000 rpm for 10 mins after 2 h reaction at 50 ℃, and then washed sequentially with ethanol and water for three times, respectively.

Preparation of nanocomposites of MPDA and TZP

MPDA particles were re-dispersed in deionized water for further use, and then TZP (MedChemExpress, USA) were added into dispersed MPDA nanoparticles above with a magnetic mixer (Thermo Scientific, USA) at 4 °C, 24 h for MPDA@TZP nanocomposites formation.

Physicochemical characterization of MPDA and MPDA@TZP nanoparticles

10 μL of suitable dilute sample solution is dropped into a copper net of amorphous carbon, followed by being dried in a dryer containing anhydrous calcium carbonate. Then the size and morphology of MPDA and MPDA@TZP nanoparticles were detected with 120 kV transmission electron microscope (TEM) (120 kV, JEM-1400 Plus). The Zeta potential and hydrodynamic size of the nanoparticles were detected by dynamic light scattering (DLS) at 25 ℃ with Zetasizer Nanopotentiometer. X-ray photoelectron spectroscopy (XPS, Thermo ESCALAB 250XI, Thermo Kalpha) was used to determine the content changes of C, H and O elements in nanoparticles.

Drug loading and encapsulation efficiency assays

FITC_TZP fluorescent polypeptide was synthesized by Qiangyao Bio Tech. To confirm the loading capacity of TZP in MPDA, FITC_TZP (0.1, 0.2, 0.5, 1, 2, 5, 10, 15, 20 μg) was added into dispersed MPDA (1 mL, 100 μg/mL) at room temperature for 1–2 h, followed by centrifuging at 9000 rpm, 10 min. For the loading capacity of FITC_TZP, it was assessed with the detection of fluorescence spectral scanner (Invitrogen, Carlsbad). The drug loading capacity of TZP was calculated as the formula: (TZP mass in nanoparticle)/ (nanoparticle mass) × 100 %. The encapsulation efficiency of TZP was calculated as the formula: (TZP mass in nanoparticle)/ (total TZP mass) × 100 %.

Drug release studies in vitro

To confirm the release efficiency of TZP in MPDA@TZP, MPDA@FITC_TZP (1 mL, 100 μg/mL) was dispersed in PBS and water for 5 days, and the fluorescence intensity of FITC_TZP in the supernatant was measured, and then the release rate of FITC_TZP was calculated as the formula: (supernatant TZP mass)/ (total TZP mass) × 100 %.

Ethics statement

All experiments involving animals were conducted according to the ethical policies and procedures approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University (Approval NO. 2022000154).

Animal studies

DIO male mice are from GemPharmatech Co. LTD (Changzhou, China); db/m and db/db male mice are from Cavens Laboratory Animal Co. LTD (Changzhou, China). All mice were maintained in cages in 12 h light/dark cycles and have free access to water and food. 12-week-old C57BL/6 male mice were fed ad lib with a standard rodent chow diet and randomly distributed into four groups as follows: PBS, MPDA (10 mg/kg), MPDA@TZP (10 mg/kg@10 nmol/kg) and TZP (10 nmol/kg) twice a week for 2 weeks. For mice with DIO, 5-week-old C57BL/6 mice were provided with standard rodent chow diet ad lib for NCD mice or HFD (60 % of kcal from fat, D12492, Research Diets) ad lib to induce DIO mice. After 16 weeks, the mice were distributed into four groups randomly: NCD_MPDA, HFD_MPDA, HFD_TZP and HFD_MPDA@TZP. For db/db and db/m mice, 8-week-old mice were provided with a standard rodent chow diet ad libitum and randomly distributed into three groups as follows: db/m_MPDA, db/db_MPDA and db/db_MPDA@TZP. No blinding was done. Animals received subcutaneous iWAT local injections twice a week for 4 weeks and sacrificed after euthanasia by sodium pentobarbital. The body weight, fed/fasting plasma insulin, fed/fasting blood glucose levels, fed plasma leptin and food intake were analyzed. Plasma samples were harvested in EDTA-coated tubes. Tissues were harvested with snap frozen in liquid nitrogen. Metabolic cage experiments were conducted at Guangzhou University of Chinese Medicine Animal Core. The Comprehensive Lab Animal Monitoring System (CLAMS, Columbus Instruments) was used to measure energy expenditure, O2 consumption, total locomotor activity, and food intake.

Distribution analysis of TZP in vivo

The method of MPDA@FITC_TZP nanocomposites preparation was same to MPDA@TZP above. MPDA@FITC_TZP was injected via iWAT local injection and then the tissues were isolated. The fluorescence intensity of FITC_TZP in tissue was detected at intervals with Living Image 4.4.

Glucose and insulin tolerance tests

For the GTT experiment, mice were fasted overnight (16 h) and administrated with 1.5 g/kg body weight glucose via intraperitoneal injection. The concentrations of blood glucose were detected at 0, 15, 30, 60, 90, and 120 min after glucose (G7041, Sigma-Aldrich) injection. For the ITT measurement, mice were fasted for 4 hrs and received an intraperitoneal injection of insulin (0.75 U/kg body weight) (Eli Lilly). The concentrations of blood glucose were detected at 0, 15, 30, 60, 90, and 120 min after insulin injection.

Analysis of metabolites and circulating factors

Blood samples were harvest in tubes with EDTA-coated, kept on ice, centrifuged and then stored supernatant at − 80 °C. Plasma triglycerides, FFAs and cholesterols were detected using corresponding kits as manufacturer instructions (Mindray Chem Inc.). Insulin (Crystal Chem Inc.) and leptin (Crystal Chem Inc.) were measured by ELISA.

Histology and immunohistochemistry

For adipose tissue H&E (hematoxylin and eosin) staining, tissues were isolated and then fixed in 10 % formalin overnight. For UCP1 protein immunohistochemical analysis, unstained adipose tissue sections were incubated at 60 °C for 30 min and then rehydrated. The slides were boiling in 10 mM sodium citrate buffer (pH = 6.0) for antigen retrieval, and then were incubated sequentially with anti-UCP1 antibody (A5857, ABclonal) at 4 °C overnight, followed by peroxidase polymer anti-rabbit, and 3,3-diaminobenzidine. Then the slides were mounted (Vector Shield) and photographed with Caseviewer.

Gene expression analysis

The gene expression level was analyzed by qPCR as previously described procedure [29]. Total iWAT RNA was extracted using TRIzol (Invitrogen) according to the manufacturer manuals. The information of qPCR primers was listed in Supplementary Table S1. iWAT RNA-Seq was performed using Novaseq6000. RNA-Seq data were analyzed using DESeq2. Transcriptomic data are available upon reasonable request to the contact corresponding authors.

Immunoblotting analyses

Tissue protein lysates were pulverized by steel ball (G0103-200G, Servicebio) in a protein lysis buffer containing 50 mM Tris-HCl (pH 8.0), 1 % TritonX-100, 1 mM EDTA, 150 mM NaCl (pH 7.5) and the protease inhibitors. Protein lysates were denatured, centrifuged, run on an SDS-PAGE gel, and films were incubated with the specified antibodies. Primary antibodies used in the study were as below: p-ACC (3661, Cell Signaling), ACC (3676, Cell Signaling), p-AMPK (2531, Cell Signaling), AMPK (2532, Cell Signaling), p-STAT3 (9145, Cell Signaling), STAT3 (A19566, ABclonal), p-JAK2 (3776, Cell Signaling), JAK2 (3230, Cell Signaling), β-tubulin (AC008, ABclonal) and GAPDH (AC002, ABclonal).

Adipose tissue explants culture

iWATs were isolated from NCD and DIO mice as previously [30]. Specifically, iWATs were collected and then pooled in 10 cm dishes with 20 ml DMEM. iWAT tissues were sliced into small patches (∼4mm), followed by PBS and DMEM washes in turn. iWAT pieces were equally distributed into 6-well plates and cultures in M199 media with 1 nM insulin (I6279, Sigma), 1 nM dexamethasone for 24 h (D4902, Sigma). For iWATs from NCD mice, following treatments were PBS, leptin (1 μM) (HY-P70704A, MCE), TZP (100 nM), leptin(1 μM) + TZP (100 nM), 30 min); For iWATs from DIO mice (MPDA or 10 nM MPDA@TZP pre-treatment for 2 weeks in vivo), following treatment were PBS or leptin (1 μM) for 30 min [31]. Tissues were harvested and subjected to immunoblotting analyses.

Statistical analysis

Two-tailed Student t-test and one-way ANOVA with Turkey’s multiple comparisons test were used for statistical significance analyses. Data analysis was performed with “GraphPad Prism 10.0′′. p < 0.05 (*p < 0.05, **p < 0.01, and ***p < 0.001) was regarded as statistically significant. Data were presented as means ± SEM.

Results

Characterization of MPDA-encapsulated TZP in vitro

To confirm that the nanocomposite was successfully formed, synthesized MPDA@TZP nanocomposite was analyzed with TEM. The appearance of MPDA@TZP was smoother than that of MPDA particles alone (Fig. 1A and B). Both MPDA and MPDA@TZP nanoparticles exhibited similar diameters of approximately 200 nm. XPS analysis further confirmed that peptides were successfully loaded onto MPDA nanoparticles. The C, N, and O elements of both MPDA and MPDA@TZP were identified by the peaks of binding energy at 283, 398, and 530 eV, with additional higher peaks observed in the nanocomposites (Fig. 1C). The MPDA nanoparticles were negatively charged (approximately −25 mV). Upon combination with TZP, the charge shifted to a positive value (around + 6 mV), indicating the successful attachment of positively charged TZP to MPDA (Fig. 1D). Thus, by thoroughly stirring with MPDA particles, TZP was efficiently incorporated into the mesoporous structures of the nanoparticles through electronic interaction of hydroxyl group present on MPDA surface.

Fig. 1.

Characterization of MPDA@TZP both in vitro and in vivo. (A) Diagram showing the interaction between MPDA and TZP. (B) TEM images of MPDA and MPDA@TZP particles. (Scale bar, 200 nm). (C) Binding energy of MPDA and MPDA@TZP particles. MPDA: blue line, MPDA@TZP: red line. (D) Zeta potential of MPDA and MPDA@TZP particles. MPDA: blue column, MPDA@TZP: red column. (E) The loading capacity of TZP by MPDA. (F) The encapsulation efficiency of MPDA@TZP. (G) The releasing profiles of TZP from MPDA@TZP particles in PBS and water. PBS: purple line, water: blue line. (H-I) Tracing of FITC-labelled TZP in tissues before and after MPDA encapsulation with iWAT local injection, with quantification shown in (I) (n = 2 per group, Twelve-week-old C57BL/6J male WT mice were administered a single iWAT injection of TZP or MPDA@TZP (10 nmol/kg). (Li: Liver, He: Heart, Sp: Spleen, Pa: Pancreas, Br: Brain, Lu: Lung, Ki: Kidney, Qu: Quadriceps, iWAT: Inguinal White Adipose Tissue, eWAT: Epididymal Adipose Tissue, BAT: Brown Adipose Tissue). TZP: Blue line, MPDA@TZP: red line. Data represent mean ± SEM, *p < 0.05, ***p < 0.001, MPDA vs MPDA@TZP, unpaired student’s t test.

The porous characteristics of MPDA nanoparticles could affect the loading and release behaviors of TZP. Our experiments revealed that MPDA exhibits a relatively high loading efficiency of approximately 80 % (Fig. 1E). According to the typic absorption peak of peptide bond at 220 nm, a standard curve linking concentration versus absorbance was established. The MPDA@TZP nanocomposites showed a significant decrease in encapsulation rate after 1 h, reaching a maximum encapsulation rate of around 80 % (Fig. 1F). The release rate of TZP from MPDA nanoparticles was also investigated. Continuous TZP release over a period of 0–5 days, with a release rate reaching approximately 80 % on day 5 (Fig. 1G). Importantly, results of Fluorescein Isothiocyanate (FITC) labelled TZP showed that the fluorescence intensity increased over the first 6 h in the MPDA@TZP group, while it decreased from the beginning in the TZP alone group. This pattern reflected a sustained-release behavior of the MPDA@TZP formulation and indicated that MPDA encapsulation significantly extended the TZP functioning over time in iWAT (Fig. 1H and I). In summary, we successfully synthesized the MPDA@TZP nanocomposite and the MPDA microsphere nanoparticles demonstrated high efficiency in the loading and sustained release of TZP in iWAT.

MPDA encapsulation of TZP amplifies its weight loss effect via iWAT injection in wild-type (WT) mice

To evaluate whether MPDA encapsulation would enhance the metabolic benefits of TZP, WT mice were used and received iWAT local injections with PBS, MPDA, TZP, or MPDA@TZP. Treatment with TZP significantly reduced body weight compared with PBS or MPDA (Fig. 2A and B). Notably, the MPDA@TZP group showed a more significant decrease in body weight than TZP alone group after two weeks. Furthermore, random blood glucose levels decreased following TZP treatment, and no significant difference was observed between the MPDA@TZP and non-encapsulated TZP groups (Fig. 2C). Both iWAT and epididymal white adipose tissue (eWAT) weights were decreased upon TZP administrations (Fig. 2D–F). Interestingly, the weights of iWAT and eWAT in the MPDA@TZP group were significantly lower than those in the TZP alone group. These findings support that MPDA encapsulation enhances the efficacy of TZP in promoting weight loss compared with TZP alone.

Fig. 2.

MPDA encapsulation of TZP amplifies its weight loss effect via iWAT local injection in normal chow diet (NCD) mice. Twelve-week-old C57BL/6J male WT mice were injected with TZP or MPDA@TZP (10 nmol/kg) twice a week for 2 weeks via iWAT local injection. (A-B) Curves of body weight and body weight change during the treatments. (C) Random blood glucose levels of NCD-fed mice with indicated treatments. (D) Representative iWAT pictures from mice with indicated treatments. (E-F) Tissue weights and tissue/body weight ratios were analyzed with indicated treatments. n = 6–7 per group. A-C, PBS: lines with black/circle, MPDA: lines with grey/square, TZP: lines with orange/equilateral triangle, MPDA@TZP: lines with red/ Inverted triangle. E-F, PBS: black column, MPDA: grey column, TZP: orange column, MPDA@TZP: red column. Data in A-C represent mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, MPDA vs TZP, ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, MPDA vs MPDA@TZP, ^$p < 0.05, ^$$p < 0.01, ^$$$p < 0.001, TZP vs MPDA@TZP, unpaired student’s t test; Data in E-F represent mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA with Turkey’s multiple comparisons test.

MPDA encapsulation of TZP amplifies the weight loss via iWAT local injection in DIO mice

To further explore the chronic effect of MPDA-encapsulated TZP on metabolic profiles under disease setting, we performed the chronic studies in DIO mice. The results demonstrated that administrations of TZP significantly reduced body weight in DIO mice, with a more pronounced reduction observed in the MPDA@TZP group compared with the non-encapsulated TZP group (Fig. 3B and C). Specifically, after 4 weeks of treatment, the MPDA@TZP group exhibited approximately 40 % reduction in body weight, whereas the TZP alone group exhibited an approximately 20 % reduction (Fig. 3D). As expected, food intake was significantly reduced in the MPDA@TZP group compared with the TZP alone group (Supplementary Fig. S1A). Additionally, both fasting and fed blood glucose levels decreased following TZP treatment (Fig. 3E and F). Notably, reductions in both fasting and fed plasma insulin levels were observed only in the MPDA@TZP group (Fig. 3G and H). The Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) score was also significantly lower in the MPDA@TZP group, approaching levels comparable to the normal chow diet (NCD) control group (Fig. 3I). Interestingly, TZP treatment markedly improved glucose tolerance and insulin sensitivity, showing no notable difference between the MPDA@TZP and TZP alone groups (Fig. 3J–M). Moreover, the MPDA@TZP group showed a more significant increase in energy expenditure compared with the high-fat diet (HFD) control group (Fig. 3N), as well as in total physical activity (Supplementary Fig. S1B). However, no apparent change was observed in the respiratory exchange ratio (RER) following TZP treatment (Supplementary Fig. S1C). Collectively, these results suggest that TZP treatment significantly decreases body weight, improves glucose metabolism, and enhances energy expenditure in mice with obesity, with these effects being further amplified by iWAT injection after MPDA encapsulation.

Fig. 3.

MPDA encapsulation of TZP amplifies the weight loss effect via iWAT local injection in DIO mice. Twenty-one-week-old DIO mice (HFD for sixteen week) were injected with 10 nmol/kg TZP or MPDA@TZP twice a week for 4 weeks via iWAT local injection. (A) Diagram showing the experimental design. (B) Representative images of mice with indicated treatments for 4 weeks. (C-D) Curves of body weight and body weight change. (E-F) Blood glucose levels under fed (E) and fasting (F) states were measured. (G-I) Plasma insulin levels of mice under feeding (G) and fasting (H) statuses and calculated HOMA-IR (I) were shown with indicated treatments. (J-K) Glucose intolerance tests (J) and quantification of area under curve (AUC) (K) were shown. (L-M) Insulin sensitivity tests (L) and ACU quantification (M) were shown. (N) Energy expenditure rates were shown. C-M, n = 5–8 per group; N, n = 3–5 per group. C, D, J, L, NCD_MPDA: lines with black/circle, HFD_MPDA: lines with grey/square, HFD_TZP: lines with orange/equilateral triangle, HFD_MPDA@TZP: lines with red/ Inverted triangle. E-I, K, M, N, NCD_MPDA: black column, HFD_MPDA: grey column, HFD_TZP: orange column, HFD_MPDA@TZP: red column. Data in C, D, J and L represent mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, HFD_MPDA vs HFD_TZP, ^$p < 0.05, ^$$p < 0.01, ^$$$p < 0.001, HFD_TZP vs HFD_MPDA@TZP, unpaired student’s t test; Data in E-I, K, M and N represent mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA with Turkey’s multiple comparisons test.

MPDA encapsulation of TZP promotes the reduction of adipose tissue weight via iWAT local injection in DIO mice

To study how MPDA@TZP amplifies the weight loss via iWAT local injection in DIO mice, plasma metabolites and circulating factors were subsequently analyzed. Compared with the HFD control group, 4-week MPDA@TZP treatment significantly reduced circulating triglyceride (TG) levels, free fatty acids (FFA), and total cholesterol (TC), whereas TZP alone only showed a significant reduction in TC (Fig. 4A–C). Additionally, adipose tissue weight was significantly decreased after TZP treatment, while it was significantly lower in the MPDA@TZP group compared with TZP alone (Fig. 4D and Supplementary Fig. S2). However, there was no difference in liver and quadriceps muscle (Quad) ratio after TZP treatment (Fig. 4E). Besides, the adipocyte sizes in iWAT of the MPDA@TZP group, but not the TZP alone group, were smaller compared with those in the HFD control group (Fig. 4F–H). Immunohistochemical staining revealed apparent induction of UCP1 protein level following MPDA@TZP treatment compared to HFD control (Fig. 4I). Besides, no obvious toxicity were observed in the injected WAT tissues and systemic levels from mice treated with MPDA@TZP or MPDA for 4 weeks, as indicated by H&E staining, Sirus Red staining, serum ALT and AST levels and hepatic H&E staining (Supplementary Fig. S3). Collectively, these results indicate that MPDA@TZP not only attenuated adipose tissue weight and adipocyte hypertrophy, but also improved systemic lipid profile that were superior to those achieved with TZP alone.

Fig. 4.

MPDA@TZP promotes the reduction of adipose tissue weight via iWAT local injection in DIO mice. Twenty-one-week-old DIO mice (HFD for sixteen week) were injected with 10 nmol/kg TZP or MPDA@TZP twice a week for 4 weeks via iWAT local injection. (A-C) Plasma levels of TG (A), FFA (B) and TC (C) levels were measured after 4 weeks of treatments. (D) Tissue/body weight ratios of WAT and BAT were analyzed. (E) Tissue/body weight ratios of livers and quad muscles were analyzed. (F) Representative images of H&E staining for iWAT, eWAT and BAT tissues. (Scale bar, 100 μm). (G-H) Quantification of adipocyte areas for iWAT (G) and eWAT (H) by H&E staining. (I) UCP1 immunostaining of iWAT and BAT sections. (Scale bar, 100 μm). A-E, n = 5–8 per group; G-H, n = 3 per group. A-E, G, H, NCD_MPDA: black column, HFD_MPDA: grey column, HFD_TZP: orange column, HFD_MPDA@TZP: red column. G-H, NCD_MPDA: lines with black/circle, HFD_MPDA: lines with grey/square, HFD_TZP: lines with orange/equilateral triangle, HFD_MPDA@TZP: lines with red/ Inverted triangle. Data represent mean ± SEM *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA with Turkey’s multiple comparisons test.

Improved lipid metabolism by MPDA-encapsulated TZP is associated with elevated BCAA catabolism and attenuated leptin resistance in iWAT

To elucidate the mechanisms on how MPDA encapsulated TZP improves weight loss and lipid metabolism, transcriptomic analysis of iWAT in NCD, HFD control, and HFD_MPDA@TZP groups were performed. A total of 926 and 530 overlapped regulated differentially expressed genes (DEGs) were identified between the MPDA@TZP and the HFD control group (Supplementary Fig. S4A). KEGG analysis indicated that the upregulated genes in MPDA@TZP was significantly enriched in pathways involved in the catabolism of branched-chain amino acids (BCAAs), fatty acid metabolism, mitochondrial substrate transport, and electron transport activity (Fig. 5A and B). However, the genes involved in lipolysis did not change after MPDA@TZP treatment (Supplementary Fig. S4B). Given that adipose tissue is an endocrine organ, the adipokine pathways were analyzed. The top four genes within the adipokine pathway were identified, with leptin being the most downregulated, consistent with decreased plasma leptin levels (Fig. 5C and D). The quantitative PCR (qPCR) analyses confirmed that MPDA@TZP iWAT injection induced the expression of genes related to fatty acid oxidation, mitochondrial metabolism, and BCAA catabolism in iWAT (Fig. 5E). Furthermore, the JAK2/STAT3 and AMPK/ACC signaling pathways in iWAT were examined, which are downstream of LepR signaling and involved in lipid metabolism. Phosphorylation levels of AMPK and ACC, but not JAK2 and STAT3, were decreased in the HFD group compared to NCD controls (Fig. 5F and G). However, the phosphorylation levels of AMPK, ACC, JAK2, and STAT3 were upregulated following TZP treatment. These results support that MPDA@TZP injection in iWAT improves body weight loss and leptin resistance associated with improved metabolic pathways including lipid catabolism, mitochondria activity, and BCAA catabolism.

Fig. 5.

MPDA@TZP enhances BCAA catabolism and attenuates leptin resistance in DIO mice via iWAT local injection. Twenty-one-week-old DIO mice (HFD for sixteen week) were injected with 10 nmol/kg TZP or MPDA@TZP twice a week for 4 weeks via iWAT local injection. (A) KEGG analysis for overlapped genes down-regulated in HFD_MPDA vs NCD_MPDA and up-regulated in HFD_MPDA@TZP vs HFD_MPDA up. (B) Clusters of DEGs involved in fatty acid oxidation, mitochondrial metabolism and BCAA catabolism were identified in iWAT after MPDA@TZP treatment. (C) KEGG analysis of DEGs up-regulated in HFD_MPDA vs NCD_MPDA while down-regulated in HFD_MPDA@TZP vs HFD_MPDA (DEG-overlap1) and down-regulated in HFD_MPDA vs NCD_MPDA while up-regulated in HFD_MPDA@TZP vs HFD_MPDA (DEG-overlap2). (D) Plasma leptin levels were measured by ELISA (n = 5–7 per group). (E) qPCR analysis of gene expression levels from iWAT identified in (B) (n = 5–6 per group). (F-G) Immunoblots of iWAT lysates for LepR downstream pathways (F) and quantification were shown (G) (n = 3 per group). D, E, G, NCD_MPDA: black column, HFD_MPDA: grey column, HFD_TZP: orange column, HFD_MPDA@TZP: red column. Data represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA with Turkey’s multiple comparisons test.

TZP activates leptin signaling and improves leptin resistance in iWAT

To further explore whether the beneficial effects of TZP was dependent on local LepR signaling in adipose tissue, iWAT explants isolated from NCD-fed WT mice were treated with leptin and/or TZP (Fig. 6A). Both leptin and TZP treatment alone induced the activation of LepR signaling pathway, as indicated by increased phosphorylation levels of AMPK, ACC, JAK2, and STAT3, whereas the combined treatment showed synergistically increased phosphorylation levels of these proteins compared with either leptin or TZP treatment alone (Fig. 6B and C). Further, iWAT explants isolated from DIO mice were used to investigate the effect of leptin and TZP on LepR signaling (Fig. 6D). As expected, leptin treatment failed to induce LepR signaling in cultured explants, confirming the leptin resistance of iWAT under HFD-feeding (Fig. 6E and F). Interestingly, MPDA@TZP pre-treatment for 2 weeks (twice per week) not only activated LepR signaling of iWAT explants in DIO mice compared with vehicle treatment, but also reversed leptin resistance in iWAT as indicated by the treatment with MPDA@TZP (in vivo) followed by leptin stimulation (ex vivo) (Fig. 6E and F). These results suggest that TZP could activate leptin signaling and attenuate leptin resistance in iWAT induced by HFD-feeding.

Fig. 6.

TZP activates leptin signaling and improves leptin resistance in iWAT of HFD-fed mice ex vivo. (A) Diagram of the experimental design in iWAT explants from NCD mice. (B-C) Immunoblots of proteins downstream of leptin receptor signaling from iWAT lysates of NCD mice (B) and the quantifications (C). (D) Diagram of the experimental design for treating cultured iWAT explants extracted from DIO mice with or without MPDA@TZP pre-treatment. (E-F) Immunoblots of proteins downstream of leptin receptor signaling from leptin or control treated iWAT explants (E) and the quantifications (F). n = 3 per group. C, F, PBS: black column, leptin: sky blue column, MPDA@TZP: blue column, MPDA@TZP + leptin: red column. Data represent mean ± SEM., *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA with Turkey’s multiple comparisons test.

MPDA-encapsulated TZP improves metabolic phenotypes via iWAT local injection in db/db mice

The results above demonstrated that MPDA-encapsulated TZP was more effective in inducing weight loss than TZP alone, associated with improved LepR signaling. Thus, MPDA@TZP was used to further verify the function and mechanism of TZP in body weight loss under obesity in db/db mice, which were LepR deficient (Fig. 7A). Our results showed that iWAT injection of MPDA@TZP after 4 weeks led to reduced body weight in db/db mice (Fig. 7B and C), as well as significantly decreased food intake compared with MPDA treatment (Supplementary Fig. S5A). Interestingly, only approximately 5 % weight loss was observed in MPDA@TZP group comparing with MPDA treatment, which was apparently less effective than the ∼ 40 % reduction of body weight observed in DIO mice (Fig. 7D and E).

Fig. 7.

MPDA@TZP improves metabolic phenotypes via iWAT local injection in db/db mice. Eight-week-old db/db and db/m mice were injected with 10 nmol/kg TZP or MPDA@TZP twice a week for 4 weeks via iWAT local injection. (A) Diagram of the experimental design. (B) Representative images of treated mice. (C-D) Curves of body weight and body weight change during the treatments. (E) Comparison of body weight change ratios in db/db and DIO mice after MPDA@TZP treatment. (F-G) Blood glucose levels under feeding (F) and fasting (G) states. (H-J) Plasma insulin levels under feeding (H) and fasting (I) states, and HOMA-IR scores were calculated (J). (K-L) Glucose tolerance tests (K) and calculated AUCs (L). (M) Comparison of the glucose lowing effects of MPDA@TZP in db/db and DIO mice. (N-O) Insulin tolerance tests (N) and calculated AUCs (O). (P) Comparison of the insulin sensitivity after MPDA@TZP treatment in db/db and DIO mice. (Q) Energy expenditure rates after MPDA@TZP treatment in db/db mice. C-J, n = 6–10 per group; K, L, N and O, n = 4–5 per group; M and P, n = 4–8 per group; Q, n = 4–5 per group. C, D, K, N, db/m_MPDA: lines with black/circle, db/db_MPDA: lines with blue/square, db/db_MPDA: lines with red/equilateral triangle. F-J, L, O and Q, db/m_MPDA: black column, db/db_MPDA: blue colum, db/db_MPDA@TZP: red column. E, M and P, db/db_MPDA: grey column, db/db_MPDA@TZP: orange column, HFD_MPDA: black column, HFD_MPDA@TZP: red column. Data in C, D, K and N represent mean ± SEM, db/db_MPDA vs db/db_MPDA@TZP, *p < 0.05, **p < 0.01, ***p < 0.001, unpaired student’s t test; Data in E-J, L-M and O-Q represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA with Turkey’s multiple comparisons test.

Both fasting/fed insulin and blood glucose levels, as well as calculated HOMA-IR, were significantly decreased in MPDA@TZP group compared with the MPDA group (Fig. 7F–J). Consistent with the findings above in DIO mice, MPDA@TZP treatment not only markedly improved glucose tolerance (Fig. 7K–M), but also enhanced insulin sensitivity (Fig. 7N–P). However, there were no differences in the energy expenditure rate (Fig. 7Q and Supplementary Fig. S5B) and the physical activity (Supplementary Fig. S5C) after MPDA@TZP treatment. Interestingly, the RER in MPDA@TZP-treated mice was significantly elevated, indicating a preference towards carbohydrate substrate utilization (Supplementary Fig. S5D). Collectively, these results indicate that MPDA@TZP treatment was less effective in LepR-deficient mice compared with obese WT mice.

MPDA-encapsulated TZP enhances BCAA catabolism in iWAT through activating LepR signaling

Next, metabolic profiles of db/db mice were analyzed after MPDA@TZP treatment. Results showed that there was no significant difference in circulating TG and TC levels after MPDA@TZP treatment, excepted for reduced plasma FFA levels (Fig. 8A–C). Liver weight and the liver/body weight ratio were reduced in the MPDA@TZP group than MPDA control, but adipose tissue weights remained comparable (Fig. 8D and Supplementary Fig. S6A). Similarly, the sizes of adipocyte were comparable between the MPDA@TZP and db/db control groups (Fig. 8E and Fig. Supplementary S6B-C). Immunohistochemical staining indicated that UCP1 protein levels were also comparable between MPDA@TZP and db/db control groups (Fig. 8F). Moreover, iWAT RNA-Seq analysis revealed that leptin signaling and genes governing fatty-acid oxidation, mitochondrial metabolism, and BCAA catabolism were only modestly changed by MPDA@TZP treatment in db/db mice comparing with the marked alterations in DIO mice. (Fig. 8G). Consistently, there was no difference in plasma leptin levels (Fig. 8H). qPCR analysis confirmed that the expression levels of genes related to fatty acid oxidation, mitochondrial metabolism, and BCAA catabolism in iWAT from MPDA@TZP-treated mice were comparable to that of db/db control (Fig. 8I). In addition, the phosphorylation levels of AMPK and ACC were lower in db/db mice than in db/m control, yet MPDA@TZP treatment did not reversed the phosphorylation levels of AMPK, ACC, JAK2, and STAT3 (Fig. 8J and K). These results suggest that the effects of iWAT injected MPDA@TZP on body weight, along with enhanced lipid oxidation, mitochondrial activity, and BCAA catabolism were at least partially dependent on LepR signaling.

Fig. 8.

Enhanced BCAA metabolism in iWAT by MPDA@TZP via iWAT local injection is dependent on LepR signaling. Eight-week-old db/db and db/m mice were injected with 10 nmol/kg TZP or MPDA@TZP twice a week for 4 weeks via iWAT local injection. (A-C) Plasma levels of TG (A), FFA (B) and TC (C) after 4 weeks of treatment. (D) Tissue/body weight ratio analysis. (E) Representative H&E staining images of iWAT, eWAT and BAT sections. (Scale bar, 100 μm). (F) UCP1 immunostaining of iWAT and BAT sections. (Scale bar, 100 μm). (G) Transcriptomic analysis of gene expression levels involved in fatty acid oxidation, mitochondrial metabolism and BCAA catabolism in db/db and DIO mice. (H) Plasma leptin levels measured by ELISA. (I) qPCR analysis gene expression levels in iWAT of mice with indicated treatments (n = 4–5 per group). (J-K) Immunoblots of iWAT lysates (J) and quantifications (K) were shown (n = 3 per group). A-D and H, n = 6–10 per group. A-D, I and K, db/m_MPDA: black column, db/db_MPDA: blue colum, db/db_MPDA@TZP: red column. Data represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA with Turkey’s multiple comparisons test.

Discussion

Multi-targeted pharmacotherapy is one of the most promising next-generation therapies for metabolic diseases [32,33]. Clinical trials on TZP have indicated its striking impact on glycemic control and weight loss in patients with T2DM and/or obesity [34]. However, delivery strategies for those drugs to achieve better efficacy remain largely unexplored [23,35].

In this study, TZP was encapsulated with MPDA and locally injected into the iWAT of mice twice a week to examine its systematic effect with relatively lower dosing frequency [21]. Compared with non-capsulated TZP, MPDA-encapsulated TZP prolonged the iWAT retention with more dramatic body-weight reduction and comparable glucose lowering effect. Transcriptomic analysis of iWAT tissues from DIO mice showed decreased leptin levels alongside activated BCAA catabolism and lipid metabolism with MPDA@TZP treatment compared with HFD control. Furthermore, TZP not only activated LepR signaling, but also attenuated leptin resistance in the ex vivo iWAT explants from DIO mice. In db/db mice with LepR deficiency, iWAT local injection of MPDA@TZP significantly improved metabolic profiles; however, the decreases in body weight and the elevations in energy expenditure were far less pronounced than those observed in DIO mice. These data support that MPDA-encapsulated TZP localized in iWAT reduced fat mass in a LepR dependent manner and suggest that TZP together with nano-technology would enable precision therapies for reducing either systemic or local fat storage in patients with obesity.

Both GLP-1R and GIPR signaling pathways are important for metabolic regulation [9,[15], [16], [17]]. Accumulating clinical studies have indicated that GLP-1RAs have positive effects on obesity, nonalcoholic fatty liver disease, diabetes, cardiovascular disease, etc. [[36], [37], [38]]. The decrease of WAT mass is one of major goals of GLP-1RAs, GIPRAs and multiple-target agonists. However, whether and how those agonists could directly target WAT to enhance their beneficial effects in glucose and lipid metabolism remain inconclusive [[39], [40], [41], [42]]. Importantly, GLP-1 was shown to promote thermogenesis and adipocyte lipolysis [43], and long-term treatment of GLP-1RA could activate thermogenesis in adipose tissue in both mice and human [44,45]. In addition, GLP-1RAs promoted preadipocytes proliferation and adipocyte differentiation [46]. In contrast, it has been shown that GIPR was more widely expressed in WATs and BATs [[12], [13], [14]]. A recent study also demonstrated that GIPR was expressed in adipocytes from both mouse and human, and the function of TZP on adipocytes was mostly dependent on the GIPR agonism with improved glucose and lipid storage as well as lipid outflow by both systemic and local efficacy [39]. Consistently, we confirmed the efficacy of TZP on adipose tissue in improving lipid and glucose metabolism both locally and systematically in DIO mice. However, how GLP-1R and/or GIPR function in mediating the effect of TZP on adipose tissue in our study still needs to be further explored.

Leptin plays critical roles in the metabolic regulation of tissues, including the brain, liver, muscle, adipose tissue, and kidney, among others. [[47], [48], [49]]. Fatty acid oxidation was stimulated in adipose tissue through its downstream targets, such as the AMPK/ACC and JAK2/STAT3 signaling pathways [31,50]. Clinical trials have shown that TZP at 15 mg per week promoted dramatic weight loss of about 20 % in 56.7 % of patients with obesity [22]. More interestingly, prior studies have reported that GLP-1RAs exert different effects on body weight across distinct obese animal models. In Zucker rats lacking leptin receptors, GLP-1 induced appetite inhibition and weight loss were abolished [51]. Similarly, a GLP-1R agonist exenatide treatment led to substantial decrease in body weight in DIO mice, whereas no such change was observed in ob/ob mice [52]. Liraglutide, another GLP-1R agonist, induced an approximately 15 % reduction in body weight in DIO mice, but only a 1 % decrease in db/db mice [53]. These findings indicate that the effects of TZP on body weight reduction may be associated with LepR signaling pathways. Consistently, transcriptomic profiling of iWAT from MPDA@TZP-treated DIO mice identified that leptin as the most dramatically down-regulated gene within the adipokine pathway (Fig. 5). Remarkably, ex vivo TZP treatment of iWAT explants from HFD-fed mice alleviated leptin resistance, as evidenced by restored the AMPK/ACC1 and JAK2/STAT3 signaling pathways (Fig. 6). Consequently, the weight loss in DIO mice was more dramatic than that in db/db mice after MPDA@TZP treatment (Fig. 7, Fig. 8). These findings confirm that LepR signaling is involved in body weight reduction effects of TZP treatment.

Recent reports showed strong correlations among elevated levels of BCAAs/BCKAs, obesity, systemic IR and T2DM [54,55]. This indicates that the disorders of BCAA metabolism could be causal role in obesity and related diseases [54]. In fact, due to the reduced expression of enzymes associated with BCAA metabolism, the disruption of BCAA breakdown in adipose tissue leads to high levels of circulating BCAAs [56,57]. As a result, in the adipose tissue of obesity models, BCAA catabolism activation decreases plasma BCAAs and their product BCKAs levels to alleviate obesity [58,59]. Our study demonstrated that the improvement of TZP on obesity was partially due to increased expression of the enzymes involved in BCAA catabolism in iWAT (Fig. 5). Specifically, TZP significantly induced the production of BCAT2 and BCKDH, rate-limiting enzymes that catalyze the first two steps of BCAA breakdown, together with the downstream genes related to the BCAA catabolism signaling pathway in iWAT. These results lead to the intriguing hypothesis that TZP may decrease body weight in patients by promoting the catabolism of BCAAs in iWAT.

MPDA is an ideal drug carrier with simplified preparation, high loading capacity, strong adhesiveness, and well-documented biocompatibility, which has been widely used in preclinical trials [60]. We proposed the MPDA encapsulation and iWAT local injection could increase the local concentration of TZP in WAT with prolonged release, and reduce the circulation level of TZP, aiming to enhance the local effect of TZP to improve WAT and whole-body metabolisms. Indeed, as it has been reported that the extracellular matrix (ECM) of WAT were negatively charged, positively charged materials could show targeted enrichment in WAT [61]; Consistently, MPDA encapsulation of TZP, which showed positive charge, increased retention time of TZP in iWAT (Fig. 1, Fig. 2). However, it is not sufficient to exclude the possibility that locally injected TZP could be released into the circulation to improve metabolism through hypothalamic GLP-1R/GIPR [62,63]. Indeed, improvements in glucose tolerance and insulin sensitivity were comparable between MPDA@TZP and TZP group (Fig. 2, Fig. 3). Clinical trials have suggested that treatment with TZP led to a notable decrease in liver fat (LF) and visceral adipose tissue (VAT) weight, whereas it caused an increase in abdominal subcutaneous adipose tissue (aSAT) weight [64]. By contrast, compared to TZP alone, MPDA@TZP treatment induced reductions in both iWAT and eWAT mass, indicating that selective enhancement of iWAT metabolism contributes, at least partially, to the metabolic improvements observed in DIO mice (Fig. 3, Fig. 4). Therefore, MPDA encapsulation augments the local efficacy of TZP in iWAT and offers a promising route for precision treatment of obesity and related metabolic disorders.

Regarding patient compliance, body weight rebound after treatment discontinuation, and gastrointestinal adverse effects associated with GLP-1RA therapies, enormous efforts have been made to understand the mechanisms of single, double, and even triple targets, as well as to improve delivery strategies [23,35]. Given that WAT mass is crucial to obesity, targeting WAT and/or BAT is a promising strategy to improve the therapeutic effect of medications [11]. Recent study showed that with local delivery systems, bioactive peptides (as browning reagents) could be restricted to the treated area, and thus could expect to be minimized their potential side effects on other tissues [65]. In this study, iWAT local injection was beneficial to increase local TZP concentration, which could reduce systemic side effects. In addition, the combination of TZP with MPDA encapsulation further increased the retention time in iWAT, which could not only reduce the dosing frequency but also achieve preferable therapeutic efficacy (Fig. 1, Fig. 2), thereby potentially improving patient compliance in clinical practice. Clinically, TZP reduces lean mass/body weight ratio [22], whereas in our study, the quad/body weight ratio was comparable between HFD control and MPDA@TZP groups (Fig. 4). Besides, although our data showed that no remarkable systemic toxicity were observed after MPDA@TZP nor MPDA alone showed after 4 weeks of treatment, including liver toxicity or iWAT fibrosis or inflammation at the injected site (Supplementary Fig. S4), risks with long-term nanocarrier retention in iWAT would need to be investigated in future to warrant the translation of this technology. Thus, this localized delivery strategy might provide an alternative for fat reduction clinically, with precise tissue targeting, drug dosing and frequency reduction, and efficacy improvement [26]. To the best of our knowledge, this is the first study to demonstrate the relatively restricted local effect of TZP treatment on the metabolic profiles, providing novel insights for future strategies to improve metabolic outcomes with precision medicine.

Collectively, our findings provide improved understanding of how TZP directly regulates metabolism in specific tissues (Fig. 9). Additionally, the functions of TZP and other polypeptides could be further improved with the aid of nanomaterials, thereby guiding their clinical applications in the future.

Fig. 9.

Retention of TZP in iWAT by MPDA encapsulation amplifies its effect on weight loss is associated with enhanced lipid catabolism, mitochondrial metabolism and elevated BCAAs catabolism via LepR signaling.

Ethics statement

All experiments involving animals were conducted according to the ethical policies and procedures approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University (Approval NO. 2022000154).

Funding declaration

This work was supported by National Key R&D program of China (2023YFF0724200 to X Shuai, Z Xiao & G Shi), National Natural Science Foundation of China (82300965 to M Li, 82,470,891 to J Peng, 82,070,811 to G Shi), Postdoctoral Fellowship Project (2024 M750625 to M Li), Guangdong Basic and Applied Basic Research Foundation (2025A1515011240 to J Peng, 2024A1515012501 to G Shi), Guangzhou Municipal Science and Technology Project (202201020497 and 2024A04J6567 to G Shi).

Data availability

Original raw data is available from the corresponding author upon reasonable request.

Compliance with ethics requirements

All Institutional and National Guidelines for the care and use of animals (fisheries) were followed.

CRediT authorship contribution statement

Lin Mi: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Writing – original draft. Tan Li: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Project administration. Xiaoxin Xiang: Data curation, Formal analysis, Investigation, Methodology, Validation. Yimin Zhou: Data curation, Formal analysis, Investigation, Methodology, Validation. Na Xiong: Data curation, Formal analysis, Investigation, Methodology. Yanyu Chen: Data curation, Formal analysis, Investigation, Methodology. Jiaoting Chen: Data curation, Investigation, Methodology. Sijia Shang: Data curation, Investigation, Methodology. Shumeng Chen: Writing – review & editing. Wai W. Cheung: Methodology, Writing – review & editing. Zecong Xiao: Conceptualization, Project administration. Yanming Chen: Conceptualization, Project administration. Jiahai Wang: Conceptualization, Project administration. Jun Peng: Funding acquisition, Investigation, Methodology, Project administration, Formal analysis, Writing – review & editing. Xintao Shuai: Funding acquisition, Investigation, Methodology, Project administration, Formal analysis, Writing – review & editing. Guojun Shi: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Writing – original draft, Writing – review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary material

The following are the Supplementary data to this article: