Pre-clinical studies show anti-diabetic effect of INSPARIN, a protein derived from the E4orf1 gene of a human adenovirus

Abstract

Background

Human adenovirus Ad36-derived protein, termed INSPARIN, up-regulates cellular glucose uptake through insulin-independent cell signaling. Transgenic or viral vector mediated delivery of INSPARIN exhibits significant anti-diabetic potential in mice. To translate these findings for clinical use, this preclinical study determined the short- and long-term effectiveness of nano-liposome-mediated delivery of INSPARIN to improve glucose metabolism in cell and animal models.

Methods

Void or INSPARIN Nanoparticles (NP) composed of soy-phosphatidylcholine were prepared freshly before use. Glucose uptake was measured in 3T3-L1, C2C12, and HepG2 cells following 72 h treatment with void or INSPARIN NP using a [³H]-2-deoxyglucose uptake assay. Male C57BL/6J mice fed a 45% high-fat diet received single or repeated subcutaneous injections of INSPARIN NP, followed by oral glucose tolerance tests, serum insulin, and HbA1c measurements. Biodistribution was assessed by DiD-labeled nanoparticle imaging, and long-term safety was evaluated by histopathology of major organs.

Results

INSPARIN NP upregulated glucose uptake in preadipocytes, hepatocytes, and myoblasts. In mice, INSPARIN NP delivered the drug to the liver when administered intravenously and to inguinal adipose tissue when administered subcutaneously. Single subcutaneous administration of INSPARIN NP promoted faster blood glucose clearance in a dose-dependent manner, but without leading to hypoglycemia. Daily subcutaneous administration of INSPARIN NP for seven weeks significantly reduced HbA1c levels despite continued high fat diet, and without any adverse effects on major organs studied.

Conclusions

These findings support suitability of nano-liposome mediated subcutaneous delivery of INSPARIN in clinical trials for treating diabetes.

Introduction

Obesity and type 2 diabetes mellitus (T2DM) share several metabolic dysregulations, including insulin resistance [1], which is a complex condition characterized by reduced sensitivity to insulin-induced glucose disposal, contributing to chronic hyperglycemia in T2DM [2]. Most medications for T2DM target the insulin signaling pathway, even though this pathway, particularly the proximal insulin signaling (comprising of insulin receptor and insulin receptor substrates) is often impaired in T2DM [3]. Therefore, improving hyperglycemia without targeting insulin signaling may be more effective in people with obesity or diabetes. We reported that adenovirus 36-derived E4orf1 protein INSPARIN shows insulin-independent anti-diabetic properties even under conditions of impaired proximal insulin receptor signaling or insulin resistance [4]. INSPARIN reduces tyrosine phosphorylation of insulin receptor substrate (IRS) and increases serine phosphorylation, thereby reducing proximal insulin signaling. Instead, it promotes the distal insulin signaling by engaging the Ras→PI3K→Akt→GLUT4 pathway to enhance cellular glucose uptake while bypassing proximal insulin-receptor/IRS signaling [5]. In adipocytes and myotubes, this distal activation promotes glucose transport, thereby increasing glucose uptake In hepatocytes, INSPARIN primarily reduces hepatic glucose output and improves hepatic metabolic handling [6]. An attractive feature of INSPARIN is its ability to promote quicker blood glucose clearance in response to a glucose bolus, even in the presence of a high-fat diet (HFD) and independent of insulin [7, 8]. Transgenic or vector-mediated expression of INSPARIN improves glucose disposal in HFD-induced mouse models of T2DM [7–11].

To clinically harness the beneficial effects of INSPARIN demonstrated by these proof-of-concept studies, a drug delivery system appropriate for humans is needed. Hence, we developed a nano-liposome-based delivery system for INSPARIN. Glutathione-S-transferase (GST)-tagged INSPARIN was encapsulated in soy-phosphatidylcholine nanoparticles (NP). The NP were characterized and tested in 3T3-L1 preadipocytes. The cells were treated with free INSPARIN, INSPARIN containing NP (INSPARIN NP), or INSPARIN-free NP (void NP). The presence of INSPARIN in cells was confirmed at 24, 48, and 72 h. At 72 h after exposure, INSPARIN NP significantly decreased pTyr-IRS yet increased p-AKT protein abundance, membrane translocation of glucose transporter GLUT4, and glucose uptake compared with cells treated with void NP. Free INSPARIN (without NP) had no effect [12]. INSPARIN NP reduces tyr-phosphorylation of IRS1, indicating downregulation of insulin signaling. Yet, it upregulates downstream signaling, including pAKT and Glut4. This supports our earlier findings that INSPARIN upregulates cellular glucose uptake independent of proximal insulin signaling. Overall, this study showed that nano-liposomes successfully traffic INSPARIN inside a cell and release it, which leads to greater glucose uptake in preadipocytes [12] and demonstrated suitability of the nano-liposomes for preclinical studies of INSPARIN.

As described below, our experiments suggest that INSPARIN NP could have significant potential therapeutic application in humans with some advantages over many of the current T2DM medications. Overall, our work provides a foundation for the next step of developing INSPARIN NP for clinical studies in humans.

Methods

Nanoparticle preparation

Nanoparticles were prepared on the same day as treatment. They were composed exclusively of soy-phosphatidylcholine (Soy L-α-PC) with no additional lipids or surface modifications. The L-α-PC stock solution was prepared by dissolving 600 mg of Soy L-α-PC in 40 ml of 100% ethanol (15 µg/µl). For each 1 ml preparation, 200 µl of Soy L-α-PC stock (15 µg/µl; 3 mg total) was combined with 1 ml of INSPARIN protein solution (0.5 µg/µl; 500 µg total; Proteintech, Ag19996; GST-tagged), corresponding to a lipid : protein ratio of ~6:1 (w/w). Following ethanol evaporation of Soy L-α-PC using a nitrogen evaporator for 8–10 min, either 1 ml of INSPARIN or 1 ml of deionized water was added. The mixture was extruded using an Avanti MiniExtruder sequentially through 200 nm and then 50 nm polycarbonate filters (11 passes each), concentrated by centrifugal filtration (100 kDa cutoff; centrifuged for 25–30 min at 10,000 × g), and resuspended in 1× PBS (for in vitro studies) or saline (for in vivo studies). The encapsulation efficiency was measured using high-performance liquid chromatography (HPLC) with a C4 column (ACCUCORE 150-C4, 150 × 2.1 mm, 2.6 µm). Encapsulation efficiency was determined once at the beginning of the study and those values were subsequently used to estimate encapsulation efficiency for all preparations. Particle size and Polydispersity Index (PDI) were determined each time using BI-MAS particle size analyzer (Brookhaven Instruments, New Hampshire, USA). The encapsulation efficiency of INSPARIN NP was 74.1%, and nanoparticles typically measured 93–113 nm with a PDI of 0.06–0.23. For 1x dose, 74.1 µg of INSPARIN was injected into each mouse.

Cell culture

3T3-L1 preadipocytes (ATCC, #CCL-173) were purchased from ATCC; vendor authenticated and free of mycoplasma at purchase. No additional testing performed in our lab. Cells were maintained in high glucose (25 mM) Dulbecco’s Modified Eagle Medium (DMEM) (ThermoFisher, #11995065), with 10% Bovine calf serum (Hyclone, #SH30072.03), and 1% antibiotic- antimycotic agent Pen-Strep (Sigma Aldrich, #A5955). C2C12 and HepG2 cells were kept in maintenance media containing high glucose (25 mM) Dulbecco’s Modified Eagle Medium (DMEM) (ThermoFisher, #11995065), 10% Bovine calf serum (Hyclone, #SH30072.03,), and 1% Pen-Strept (Sigma Aldrich, #A5955). At around 70–80% confluency, cells from each well of a six-well plate were treated with 30–50 µl of either void NP or INSPARIN NP (11.11-18.52 µg of INSPARIN) and incubated for 72 h at 37 °C. Following 72 h incubation, the cells were used to perform a glucose uptake assay as described below.

Glucose uptake assay

NP-treated cells in 6-well plates were washed twice with PBS before adding 900 µL KRP (136 mM NaCl, 4.7 mM KCl, 10 mM NaPO4, 0.9 mM CaCl2, 0.9 mM MgSO4, 0.9 mM CaCl2) with or without 100 nM insulin (Sigma Aldrich, #15500) for 20 min. One well was treated with KRP plus 100 nM cytochalasin (Sigma Aldrich, #C6762) for 20 min to subtract nonspecific glucose uptake. Fifty µL of 10x isotope solution was then added to each well for a final concentration of 100 nM cold-deoxyglucose and 0.5 µCi/mL [3H]-2- Deoxyglucose (PerkinElmer, #NEC720A250UC) for 5 min. Immediately after incubation, cells were washed with ice-cold PBS and lysed with 1 mL of 0.05% SDS at 37 °C for 30–45 min. To measure glucose uptake, 950 µL of cell lysate was added to a scintillation vial containing 3 mL of scintillation liquid and read on a PerkinElmer scintillation counter (Model No. Tri-Carb 4810TR, now part of Revvity Inc.), while the remaining 50 µL used for protein estimation via BCA assay. Radioactivity Counts Per Minute (CPM) were normalized to protein content for each well.

Animals

Unless otherwise noted, all animals were housed in the same room ≤5 mice/cage on a 12-h light/12-h dark cycle and were placed on ad-libitum HFD (45% Kcal from fat; Lab Diets) and water. The experimental procedures were approved by Institutional Animal Care and Use Committee (IACUC) at Texas Tech University, Lubbock, TX. Mice were weighed according to the study design using a digital scale (OHAUS Model CL 201). The body composition of mice was measured using the EchoMRI machine (Echo MRI LLC, Houston, TX).

For the 24 h post INSPARIN NP injection experiment, 7–9 weeks old male C57BL/6J mice (n = 15) (Jackson Laboratory, Bar Harbor, ME) were fed a 45% HFD for 5 weeks and received a single subcutaneous (SC) dose of void or INSPARIN NP (1×;74.1 µg). An OGTT was performed 24 h post-injection. Next, we repeated the same experiment by including higher doses (2×;148.2 µg, or 4×;296.4 µg) and determined glucose clearance at 24 and 48 h post-injection. The doses of 2X and 4X were administered by proportionally increasing the injection volume of the standard INSPARIN nanoparticle formulation.

For long-term daily injections, 14–16 weeks old C57BL/6J mice (n = 10) fed a 45% HFD for 8 weeks were divided into a void and a treatment INSPARIN group, matched for body weights and HbA1c. The groups received daily SC injections of void-NP or 2x of INSPARIN NP (148.2 µg), for 7-weeks (6 days/week; 43 doses). OGTT, corresponding insulin, and HbA1c were determined. 2- or 6- months after the last injection, whole-body histopathology (IDEXX BioAnalytics, Missouri, USA) was performed to determine tissue specific adverse effects if any, due to chronic NP treatment.

For long-term alternate day injections, C57BL/6J mice fed a 45% HFD for 20-weeks were divided into void NP or treatment INSPARIN groups, matched for body weight and HbA1c. The groups received injections of void NP or INSPARIN NP (2×;148.2 µg), respectively, on alternate days for 8-wks, following which, glucose clearance, and HbA1c were determined.

Investigators were not blinded to group allocation during experiments or outcome assessment. Sample sizes were determined based on prior published studies using similar mouse models and expected effect sizes; no formal statistical method was used to predetermine sample size. No animals or data points were excluded from the analysis. In all the experiments, the void group received control nanoparticles composed exclusively of soy-phosphatidylcholine (soy L-α-PC) prepared identically to INSPARIN-loaded nanoparticles, except that deionized water was substituted for the protein.

Oral glucose tolerance test (OGTT)

The OGTT was performed using an oral gavage of glucose (2.5 g/kg body weight, except for one experiment where 2 g/kg body weight was used) following a 4-h fast. During the OGTT, blood glucose levels were measured via tail bleed at baseline (0 min) and at 15, 30, 60, and 120 min after the glucose load. Blood glucose level (mg/dL) during OGTT was measured using the AlphaTrak2 glucose meter. Blood (20 μL) was also collected at these time points in EDTA-coated microvettes (Sarstedt, #16.444.100) for insulin measurements.

Serum insulin measurement

Blood sera collected during OGTT were centrifuged at 10,000 rpm for 7 mins at 4 °C and stored at −80 °C. Insulin (ng/mL) was measured using the ELISA kit (EMD Millipore, EZRMI-13 K) or Mercodia mouse insulin ELISA kit (Mercodia, 10-1247-01). The manufacturer’s instructions were followed throughout the experiment. For standard curve preparation, 4-parameter logistic curve fit was used using GraphPad Prism software (version 8).

In vivo imaging using IVIS Spectrum

DiD-labeled void NP were prepared by adding DiD’ solid; DiIC18 [5] solid (1,1’- Dioctadecyl-3,3,3’,3’-Tetramethylindodicarbocyanine, 4-Chlorobenzenesulfonate Salt) (Thermo Fisher, # D7757) in the amount of 2 mol% of Soy L-α-PC and following the same preparation method explained above. Eight mice were injected with saline (control group) or DiD-labeled NP for this experiment. For the SC injection group, 2 mice received the injection on the right inguinal fat tissue, 2 mice on the left inguinal fat tissue, and 1 mouse received an SC injection of saline. For the intravenous (IV) injection group, 2 mice were injected with DiD labeled NP, and 1 mouse received saline via the tail vein. Images were obtained 48- and 72 h post-injection. Mice were anesthetized in the isoflurane chamber before obtaining the images with an IVIS® Lumina XR imaging system.

HbA1C measurement

HbA1c was measured by using A1CNow+, Hba1c Blood Monitor w/Sampler kit by following manufacture’s recommendation (PTS Diagnostics, #3021). Though this kit is for human use, we have tested it on mice. The HbA1c values in mice strongly correlate with their fasting blood glucose. For our experiments involving HbA1c measurement in mice, we have measured the HbA1c at baseline, midpoint, and post-treatment.

Statistics

Appropriate statistical tests were used, including Student’s t tests for two-group comparisons and one-way ANOVA with Bonferroni correction for multiple group comparisons. Data are expressed as mean ± SEM. For two-group comparisons, Welch’s correction was applied where appropriate, accounting for unequal variances. Normality was not formally tested, but data distributions appeared consistent with assumptions. A p value of <0.05 was considered statistically significant.

Results

INSPARIN enhances cellular glucose uptake in 3T3-L1 preadipocytes, HepG2 hepatocytes, and C2C12 myoblasts

We previously reported that nano-liposome-mediated delivery of INSPARIN promotes cellular glucose uptake in preadipocytes [12]. Considering that liver and muscle also play essential roles in glucose disposal in the body, along with preadipocytes, we repeated the experiment with hepatocytes (HepG2) and myoblasts (C2C12). We treated 3T3-L1, C2C12, and HepG2 cells with 50 µL either void or INSPARIN NP (18.52 µg of INSPARIN per well of 6 well plates, for 3T3L1 it was 11.11 µg in 30 µL), and 72 h later, we performed glucose uptake assay. Consistent with our previous results, INSPARIN NP-treated 3T3-L1 (Fig. 1A), C2C12 (Fig. 1B), and HepG2 (Fig. 1C) cells exhibited significantly higher glucose uptake compared to void-treated cells.

Fig. 1. INSPARIN NP enhances glucose uptake in preadipocytes, myoblasts, and hepatocytes.

INSPARIN NP treatment significantly increased glucose uptake in different cell types compared to void-treated controls. Glucose uptake was measured in A 3T3-L1 preadipocytes, B C2C12 myoblasts, and C HepG2 hepatocytes after treatment with void or INSP (18.525 µg per well, n = 6/group) for 72 h. Data are presented as mean ± SEM. Statistical analysis was performed using one-way ANOVA, with groups sharing the same letter indicating no significant differences (p > 0.05). INSP denotes INSPARIN-containing nanoparticles.

Intravenous injection of NP in mice

Biodistribution of DiD-labeled NP after the intravenous (IV) injection

Using the in vivo imaging system (IVIS), the biodistribution of NP was determined in 6–8 weeks old male mice after IV injection of DiD-labeled NP. At 48 h (n = 1) or 72 h (n = 1) post IV injection (50 µl), the bioluminescence of DiD-labeled NP was more expressed at the site of the injection (tail vein) (Fig. 2A, B). Following live animal imaging with IVIS, mice were sacrificed and bioluminescence determined in individual tissues. As expected with IV injections, the liver showed the strongest expression (Fig. 2C) compared with muscle, fat depots and kidneys, indicating a significant amount of the injected NP accumulated in the liver 72 h post IV injection.

Fig. 2. Biodistribution of DiD-labeled NP and effects of INSPARIN NP on glucose metabolism.

DiD-labeled nanoparticles are localized at the injection site 48 h (A) and 72 h (B) post-IV injection, with the highest accumulation in the liver (C) compared to other tissues. INSPARIN NP-treatment via tail vein injections for 2 weeks in chow-fed mice show similar glucose clearance (D) but reduced endogenous insulin levels (E) and insulin AUC (F) during OGTT three days after the final IV injection. Data are presented as mean ± SEM; Student’s t test; p < 0.05 vs. void-treated group. Epi epididymal fat, IN inguinal fat, RP retroperitoneal fat. INSP denotes INSPARIN-containing nanoparticles.

Response to oral glucose load after IV injection of INSPARIN NP in normoglycemic mice

To determine the in vivo effect of INSPARIN in normoglycemic mice, 8-week-old C57BL/6J male mice (n = 6) on regular rodent chow diet were exposed to14.4 µg of INSPARIN NP and compared with control mice (Void NP; n = 6). Mice were exposed to a total of 4 IV injections about 4 days apart over 2 weeks. Three days after the last injection, following a 4 h fasting, a 120 min oral glucose tolerance test (OGTT) (2 g/kg body weight glucose) was conducted. Blood glucose and corresponding insulin values were determined. As anticipated, mice were normoglycemic, and the glucose clearance was not different between the two groups (Fig. 2D).

Despite similar glucose clearance, the group receiving INSPARIN NP significantly reduced endogenous insulin response (Fig. 2E) and insulin area under the curve (Fig. 2F).

Response to subcutaneous injection of INSPARIN

Biodistribution of DiD-labeled NP after the SC injection

Subcutaneous injection of DiD-labeled NP (50 µl) displayed greater accumulation of bioluminescence at the site of the SC injection and hind paws after 48 h (n = 2) (Fig. 3A), and 72 h (n = 2) (Fig. 3B) as determined by IVIS. After sacrificing the mice and collecting tissues, nano-liposomes showed greater accumulation in the inguinal fat depots after 48 h (Fig. 3A) and 72 h (Fig. 3B).

Fig. 3. Evaluation of biodistribution and glycemic response following SC injection of INSPARIN NP.

DiD-labeled nano-liposomes accumulate in the injection site, hind paws, and inguinal fat 48 h (A) and 72 h (B) post-SC injection. INSPARIN enhances glucose clearance (C) and reduces glucose AUC (D) 24 h post-SC injection in HFD-fed mice (n = 15/group). No significant differences in insulin response (E) or insulin AUC (F) between groups. Higher INSPARIN doses improve glucose clearance (G) and reduce AUC (H) 24 h post-SC injection but glucose clearance (I) and AUC (J) effects are diminished 48 h post-SC injection (n = 5/group). Data presented as mean ± SEM; Students t test and ANOVA with Bonferroni: only pairs relative to void simultaneously compared for more than two groups, p < 0.05. Epi epididymal fat, IN inguinal fat, RP retroperitoneal fat. INSP denotes INSPARIN-containing nanoparticles.

Response to oral glucose load 24 h post SC injection of INSPARIN

In normoglycemic mice, IV injection of INSPARIN NP improved insulin response to glucose load (Fig. 2F). However, IV injection is a relatively invasive drug administration route for humans. Therefore, we next tested the SC injection of INSPARIN NP in a HFD-fed mice model to further investigate the effect of INSPARIN on glycemic control. Following 5 weeks of 45% HFD, at 12–14 weeks old, animals (n = 15) received one SC injection of void NP or INSPARIN NP (74.1 µg) and 24 h post-treatment, OGTT was performed. Remarkably, a single injection of INSPARIN NP significantly decreased the area under the curve (AUC) of glucose compared to the void group (Fig. 3C, D). The two groups did not display any significant differences in insulin levels (Fig. 3E, F).

Dose-response of INSPARIN NP following SC injection (48 h)

We next performed a dose response experiment, where different doses of INSPARIN NP were tested in 12–14 weeks old mice and OGTT performed 24 h and 48 h post-injection. The aim was to examine if a higher dose would further promote blood glucose clearance and whether the effect of a single dose lasts beyond 24 h. Mice receiving INSPARIN NP were divided into three groups: 5 mice received the same treatment dose as the previous experiment (1×;74.1 µg), 5 mice were injected with double that dose 2× (148.2 µg), and 5 mice received 4× that dose (296.4 µg) of INSPARIN by manipulating the dose of nano-liposomes. Also, five mice were injected with void NP. GTT was performed 24 h and 48 h post-injection. Higher doses of INSPARIN NP (2× and 4×) significantly decreased the glucose AUC at 24 h (Fig. 3 G, H) but not after 48 h post-injection (Fig. 3 I, J).

Once daily injection of INSPARIN NP for seven weeks improves glucose clearance

C57BL/6J mice (14–16 weeks old; n = 10) fed a 45% HFD for 8-weeks received daily SC injections of void NP or 148.2 µg INSPARIN NP for 7-weeks (6 days a week; 43 doses). Baseline body weight, HbA1c, fasting glucose, or insulin were not different between the groups (Fig. 4A–D). As expected, after 7-weeks of daily injections, the INSPARIN group displayed significantly faster glucose clearance (Fig. 4E, F), however, the insulin response was not significantly different compared with void mice (Fig. 4G, H). Bodyweight and body fat% were not different between groups (Fig. 4I, J). Furthermore, comprehensive histopathological analyses performed at 1- or 6-months post-treatment revealed no treatment-related immunogenicity or organ toxicity attributable to INSPARIN NP, with all observed changes being diet- or age-related. (Supplementary Tables 1, 2, Supplementary Fig. 1).

Fig. 4. Long-term subcutaneous administration of INSPARIN NP improves glycemic control in diet-induced diabetic mice.

No differences in baseline characteristics, including body weight (A), HbA1c (B), fasting glucose (C), and fasting insulin (D), were observed between the void- and INSPARIN NP-treated groups. After 7 weeks of daily SC injections of INSPARIN NP, glucose clearance was significantly improved compared to the control group (*p < 0.05) (E, F). The insulin response during the glucose tolerance test was not significantly different between the groups (G, H). Body weight (I) and body fat percentage (J) remained unchanged between groups after treatment. Long-term INSPARIN NP injections improved HbA1c levels compared to controls (K). HbA1c levels in the INSPARIN NP-treated group were significantly lower than those in the control group at both 4 weeks (*p < 0.02) and 7 weeks (**p < 0.004) (L). Data are presented as mean ± SEM; n = 10/group, Student’s t test; p < 0.05 vs. void-treated group. INSP denotes INSPARIN-containing nanoparticles.

Long-term INSPARIN NP injections reduce HbA1c

Once daily administration of INSPARIN NP for 7 weeks improved HbA1c status along with the improvement in glucose clearance (Fig. 4K). At baseline, the HbA1c levels for both groups were 5.14%. In presence of continued HFD, HbA1c changed from 5.14% to 5.12% in 4 weeks (midpoint) and to 5.53% in 7 weeks for the control group (Fig. 4L). At these time points, HbA1c changed from 5.14% to 4.91% and to 5.01% for the INSPARIN group. HbA1c was significantly lower for the INSPARIN group compared to void controls at 4 weeks (p < 0.02) and 7 weeks (p < 0.004).

After confirming that a daily dose of INSPARIN NP could clear glucose and improve HbA1c, we next determined if alternate-day dose of INSPARIN NP will be adequate for improving glycemic control instead of the daily injections.

Control and treatment groups received alternate-day SC injections of void NP or INSPARIN NP, respectively, for 8-weeks. Glucose clearance and HbA1c were determined at baseline and post-treatment. After eight weeks, no significant differences in HbA1c were observed (data not shown).

Discussion

An overarching significance of this line of inquiry is to understand how viral proteins can influence systemic metabolism and to harness their beneficial potential for human health. Previous studies by us and others uncovered anti-diabetic potential of human adenovirus Ad36, a rather unusual source. Experimental infection of Ad36 promoted cellular glucose uptake, enhanced blood glucose clearance in animal models and reduced liver fat accumulation [13–15]. Cross-sectional and longitudinal studies showed that natural infection of Ad36 in humans is associated with better glycemic profile, and lower hepatic steatosis [16–18]. Next, to harness this potential of Ad36 in a clinically meaningful manner, in a series of experiments, we identified that the E4orf1 gene of Ad36 is responsible for its anti-diabetic and anti-hepatic steatosis potential [6, 9, 19]. These proof-of-concept studies showed that a transgenic or virus vector-mediated expression of E4orf1 gene product (termed INSPARIN) enhances blood glucose clearance in mice, even in the presence of HFD [19]. Collectively, these data strongly indicate anti-diabetic potential of INSPARIN for human treatment, provided a drug delivery approach that is suitable for human use is identified. INSPARIN is generated by Ad36 virus inside host cell and does not have a receptor for it on cell surface. Hence, the in vivo testing of INSPARIN’s effect required the delivery of INSPARIN in mice using virus-vector mediated delivery or its transgenic expression [8, 11, 19]. These INSPARIN delivery methods provide proof of concept but are not a clinically useable approach. Therefore, here we tested nano-liposome-mediated delivery, a system already in clinical use [20], for delivering INSPARIN. In vitro studies previously confirmed that nano-liposome can successfully carry and release INSPARIN intracellularly and promote cellular glucose uptake [12]. Here, we conducted experiments to provide the basis for the application to the Food and Drug Administration (FDA) to conduct human phase 1 clinical trials.

Overall, the most important findings of this study were that i) INSPARIN NP enhances glucose uptake in cells of adipose tissue, skeletal muscle, and liver, indicating its potential impact on multiple tissues involved in glucose metabolism. ii) The biodistribution studies using DiD-labeled NP demonstrated the accumulation of liposomal NP at the injection site and in the liver after IV administration. This route of administration reduced endogenous insulin response to oral glucose load, but did not improve blood glucose clearance. iii) Whereas, INSPARIN NP administered subcutaneously seems to mainly spread to inguinal adipose tissue, but not to the liver. This route of INSPARIN NP administration cleared glucose faster, indicating efficacy of the subcutaneous route of drug administration. As such, subcutaneous drug administration should be more acceptable and practical for human use compared to intravenous administration. iv) INSPARIN NP promoted blood glucose clearance in a dose-dependent manner, but without leading to hypoglycemia. The dose response study with higher doses of INSPARIN NP (2X and 4X) did not extend glucose clearance beyond 24 h post injection compared to 1X INSPARIN NP dose (74.1 µg per animal). Also, the effect of single dose of INSPARIN NP on blood glucose clearance lasted for 24 h, but not 48 h post INSPARIN NP injection. Collectively, this suggests that repeated daily INSPARIN NP administration (and not high dose) would be necessary to sustain improvement in glucose metabolism. Further, it also indicates that the effect of INSPARIN NP on glycemic control is not cumulative and should not lead to any possible drug toxicity. v) Daily INSPARIN NP administration for 7 weeks led to a gradual and significant improvement in glycemic control, as evidenced by improved glucose clearance, lower insulin requirement during GTT, and lower HbA1c levels at the end of the study. This also indicated continued efficacy of INSPARIN NP after relatively long-term use and despite continued high fat feeding. Reduction of HbA1c was perhaps the most important finding that shows potential of INSPARIN NP for treating diabetes. Whereas long term administration of INSPARIN NP on alternate days was not effective in reducing HbA1c. Despite the long-term daily INSPARIN NP administration, the whole-body histopathological analysis showed no adverse effect on any of the major organs studied, indicating the safe use of INSPARIN NP. These findings indicate the necessity of the continuous presence of INSPARIN NP to attain sustained and significant improvements in glucose.

The current study provides promising results for therapeutic potential of INSPARIN in diabetes and obesity. Next, several important aspects should be addressed, including the pharmacokinetics and pharmacodynamics of INSPARIN. Also, to fully understand the anti-diabetic potential of INSPARIN, a comparative analysis of efficacy against current anti-diabetic medications should be determined.

While INSPARIN NP improved glucose clearance and HbA1c without any adverse pathology in mice, caution is warranted when extrapolating these findings to humans. Differences in pharmacokinetics, pharmacodynamics, and immune responses in humans may influence the outcome in clinical settings. Therefore, the safety, efficacy, and long-term effects of INSPARIN NP administration in humans need to be assessed through clinical trials.

Our biodistribution studies using DiD-labeled void nanoparticles demonstrated route-dependent differences between IV and SC administration (Figs. 2 and 3), these experiments were performed in a limited number of mice following a single injection. We did not directly measure tissue levels of INSPARIN protein, and chronic dosing regimens may yield broader tissue distribution than observed here. Future studies will therefore incorporate direct measurement of INSPARIN in tissues following repeated SC delivery to more fully establish the relationship between biodistribution and glycemic efficacy.

Finally, we observed reduced HbA1c with INSPARIN NP treatment, it should be noted that these values were obtained using a human-calibrated device (A1CNow+). While this method is not specifically validated for murine hemoglobin, several prior mouse studies have reported HbA1c using A1CNow/A1CNow+ [21–23], which provides precedent for its use in exploratory contexts. Moreover, our intent in this experiment was to determine relative decrease in HbA1c of individual animals as a result of INSPARIN NP treatment, and not the exact value.

In conclusion, this study demonstrates that INSPARIN NP exhibits anti-diabetic characteristics by improving glucose uptake in cell culture models and in animal models, where it could improve glycemic control even in the presence of HFD. These findings support further studies to evaluate the potential of INSPARIN as a novel anti-diabetes agent for human trials.

Author contributions

ABS, ZF, and MN collected the data. ABS analyzed the data, prepared the graphs, and wrote the manuscript. ZF assisted with manuscript writing. VH contributed to experiment planning and execution and reviewed/edited the manuscript. NVD contributed to conceptualization, experiment planning, trouble shooting, interpretation and discussion and reviewed/edited the manuscript.

Funding

Funded partly by a grant from Insparin Therapeutics and Helen Devitt Jones Endowed Chair.

Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests

As inventor, NVD has received several patents in viral obesity and adenovirus 36, including uses for E1A, E4oRF1 gene and protein, and AKT1inhibitor, and has received past grant support from Vital Health Interventions for determining anti-diabetic properties of E4-ORF1 protein. On behalf of Drs. Dhurandhar and Hegde, Texas Tech University has applied for additional patents. NVD and VH recently received funding from Insparin Inc, which is a company created to advance the development of E4orf1 as a therapeutic agent. NVD owns shares in Insparin Ins. Other authors did not have any conflict of interest.

Ethics approval and consent to participate

All animal experiments were performed in accordance with the relevant guidelines and regulations. The experimental procedures were approved by Institutional Animal Care and Use Committee (IACUC) at Texas Tech University, Lubbock, TX. This study did not involve human participants; therefore, informed consent and consent for publication are not applicable.

Supplementary information

The online version contains supplementary material available at 10.1038/s41387-025-00406-6.