Research methodology for in vivo measurements of resting energy expenditure, daily body temperature, metabolic heat and non-viral tissue-specific gene therapy in baboons.

Abstract

A large number of studies have shown that the baboon is one of the most commonly used non-human primate (NHP) research model for the study of immunometabolic complex traits such as type 2 diabetes (T2D), insulin resistance (IR), adipose tissue dysfunction (ATD), dyslipidemia, obesity (OB) and cardiovascular disease (CVD). This paper reports on innovative technologies and advanced research strategies for energetics and translational medicine with this NHP model. This includes the following: measuring resting energy expenditure (REE) with the mobile indirect calorimeter Breezing®; monitoring daily body temperature using subcutaneously implanted data loggers; quantifying metabolic heat with veterinary infrared thermography (IRT) imaging, and non-viral non-invasive, tissue-specific ultrasound-targeted microbubble destruction (UTMD) gene-based therapy. These methods are of broad utility; for example, they may facilitate the engineering of ectopic overexpression of brown adipose tissue (BAT) mUCP-1 via UTMD-gene therapy into baboon SKM to achieve weight loss, hypophagia and immunometabolic improvement. These methods will be valuable to basic and translational research, and human clinical trials, in the areas of metabolism, cardiovascular health, and immunometabolic and infectious diseases.

Introduction

Nonhuman primates (NHPs) have long been considered crucial in the study of disease (1, 2). NHPs, thanks to phylogenetic proximity, have a high degree of genetic similarity to humans, and share many physiological mechanisms relevant to normal functioning and pathogenesis (3). Recent improvements in methodology involve open new avenues for the study of immunometabolism, adipose tissue biology and obesity (OB). Here we report on some advances developed with an NHP model, the baboon (Papio sp.), an important taxon in translational research (4, 5).

Cardiovascular, immunometabolic diseases and OB are ideal targets for gene-base therapy. Such approaches focus on the transfer of gene coding or non-coding sequences to produce critical proteins to improve immunometabolic homeostasis (6), increase energy expenditure and restore insulin sensitivity (7). Brown adipose tissue (BAT) activation has emerged as an attractive target for treatment of OB, Type 2 diabetes (T2D) and cardiovascular disease (CVD). It is the main organ of thermogenesis in humans. Its capacity to oxidize fatty acids and glucose without ATP production contributes to energy expenditure (8). Brown adipocytes have a high mitochondrial content that contains the specialized protein UCP1 that causes a proton leak across the inner membrane of the mitochondria, thereby dissipating energy in the form of metabolic heat. For our preliminary rodent work on gene therapy, SKM and BAT (9) we chose a plasmid cDNA constructs encoding a gene cocktail with BMP7/PRDM16/PGC-1a incorporated within microbubbles to be delivered to the skeletal muscle of a baboon’s thigh (10). BMP7 induces the expression of PRDM16, which promotes the development of brown adipocytes and suppresses both the white adipose tissue (WAT) and the myogenic differentiation pathway (11). It has been demonstrated that PRDM16 functions as a BAT key regulator (12) leading to the expression of another key player of the brown adipocyte differentiation pathway, PGC1a. The final cellular product highly expresses the hallmark of BAT, the brown adipocyte specific gene UCP1 (13). The delivery of therapeutic genes to SKM of NHP to engineer brown adipose tissue is highly innovative (14).

Ultrasound-targeted microbubble destruction (UTMD) is a noninvasive, non-viral means of tissue-specific gene delivery in vivo. In this process, gas-filled lipid microbubbles (typically 5 μm in diameter) loaded with genetic material are circulated throughout the body (15). When exposed to ultrasound within the microvasculature of the target tissue, the microbubbles are mechanically destroyed, releasing their contents. At the same time, transient pores form in the plasma membrane facilitating diffusion of the genetic material into the cell (sonoporation) (16). The UTMD technique has successfully driven expression of plasmid cDNA in rat and baboon pancreatic islets (17). The delivery of genes by intravenously injecting plasmid DNA encapsulated in microbubbles into the animal’s blood stream, to lyse them selectively in the microcirculation of specific organs via ultrasound, is also highly innovative (18).

A mobile indirect calorimeter called Breezing® has been developed to facilitate personalized resting energy expenditure (REE) measurements in humans (19) with results similar to the ones from the gold standard Douglas bag method (20). Differing from traditional indirect calorimetry equipment--that is bulky, expensive, and complicated to calibrate and use—and equations created to estimate REE (21), this pocket-sized indirect calorimeter measures oxygen consumption rate (VO2) and carbon dioxide production rate (VCO2) in breath with a colorimetric technology, from which REE is determined according to the Weir equation (22). It also measures respiratory quotient (RQ = VCO2/VO2), which is indicative of the source of energy used at the time of the measurement (e.g., carbohydrate vs. fat).

Core body temperature is the physical state at which the internal organs and bodily systems function at an optimal level. It is an aspect of thermoregulation, the body’s ability to control its operating temperature within a constant range. Conventional studies have shown that radiotelemetric measurement of body temperature in unrestrained NHP is capable of reporting data offering significant advantages over the traditional methods of restraint combined with rectal core temperature measurements (23). Recently, small, leadless, implantable data loggers have been developed for studies on both captive and free-ranging animals. These data loggers are subcutaneously implanted to monitor continuous daily core body temperature (24).

Thermography is a non-contact, non-invasive technique that detects surface heat emitted as infrared radiation. Heat is the primary sign of inflammation process and different disease processes affect the microcirculation of the skin. Abnormal thermal skin temperature patterns can correlate with areas of inflammation or poor blood circulation. Veterinary infrared thermography (IRT) has the potential to be a useful screening method for in vivo detection through digitally imaging with an infrared camera of animals affected by any disease that induces localized inflammation. Examples of potential veterinary applications for this imaging technique have been describe in cattle to successfully detect localized sepsis, changes in udder temperature, and soles of hooves affected by subclinical laminitis (25).

The aim of this paper is to describe research methods developed in the baboon model including REE measured with a mobile indirect calorimeter, daily temperature monitoring using data loggers, veterinary IRT imaging to measure metabolic heat, and non-viral non-invasive, tissue-specific UTMD gene-based therapy. These research resources will allow and facilitate scientists to obtain new findings on the molecular mechanism that regulates food intake, adipose tissue biology, energy expenditure, fatty-acid oxidation and insulin-glucose axis regulation.

Materials and methods

Animals.

12 adult baboons (9 baboons [5 males; 4 females] to assess REE through indirect calorimetry, 2 baboons to establish proof-of-concept that UTMD is an effective method for in vivo plasmid-based gene transfer to SKM of baboons, and 1 adult male baboon to perform UTMD-based gene therapy as a proof of concept to demonstrate feasibility) were selected from a population at the Southwest National Primate Research Center (SNPRC), Texas Biomedical Research Institute (Texas Biomed), San Antonio, TX (4). The protocol was approved by the Texas Biomed Institute Animal Care and Use Committee. Animals were baboons ( Papio hamadryas sp.) fully sexually mature, housed individually during the duration of these studies in cages with access to a light cycle from the room lights in the clinic, set to be on every day from 0600 to 1800 h. These research protocols followed all recommendations from our Texas Biomed Regulatory Compliance team to ensure compliance with applicable regulations (USDA, OLAW/PHS, FDA, and CDC) and for final reports in accordance with GLP regulations.

Dietary intake and phenotypic assessment.

The quantity of food offered to each baboon daily was based on the estimated metabolizable energy requirements for adult captive baboons. Specifically, the animals were fed a commercial pelleted diet ( Monkey diet 15%, PMI Nutrition, LLC, Brentwood, MO) targeted to meet an expected energy requirement to sustain constant body weight (BW) of 40–51 kcal/kg. The exception was a 20 Kg baboon chosen to perform the proof of concept method for UTMD gene therapy. We decided to offer this animal 80 kcal/kg/day (1,600 kcal a day) before and after gene therapy administration due to the hypothetical increased effect on energy expenditure after gene therapy. We offered 53 biscuits a day (each biscuit weights ~ 7.1 g and equals 30 kcal) for daily biscuit counting through the veterinary technician assigned. Routine anthropometric measurements (26) for body weight and waist circumference were taken. Percent body fat and composition was measured by DXA, Lunar Prodigy whole body scanner; GE Medical Systems, Madison, WI (Figure 3).

Figure 3.

Comprehensive dual-energy x-ray absorptiometry (Lunar-DXA) to measure body composition (breakdown of fat tissue and muscle mass) before and after UTMD-gene therapy. Rd = Rate of disappearance (glucose uptake) mg/min per m².

Indirect calorimetry to measure REE, VO2, VCO2 and QT through a mobile indirect calorimeter.

A protocol was developed using a mobile indirect calorimeter, (Breezing®) to facilitate personalized resting energy expenditure (REE) measurement in 9 baboons. The Breezing pocket-sized indirect calorimeter measures oxygen consumption (VO2), carbon dioxide production (VCO2) and respiratory quotient (RQ). Our method included an initial two-step sedation with telazol 5–7 mg/Kg and ketamine 10 mg/Kg to ensure the animal was fully relaxed while breathing. Animals were placed in dorsal recumbency with the head extended at a 45’ angle to ensure full extension of the airway. The animal was intubated with an endotracheal tube and a T-tube (mouthpiece) was placed onto the Breezing device. The device and the T-tube were connected with the endotracheal tube and the REE measurement was started.

Generating a NHP model for UTMD to study gene therapy and OB.

Limited pilot funds were obtained to conduct a proof of concept study to administer plasmid cDNA constructs encoding BMP7/PRDM16/PGC-1a (Figure 1C) incorporated within microbubbles and intravenously delivered into the left thigh of one adult baboon. The right thigh of the same baboon was used as a control. An ultrasound transducer was directed to the baboon quadriceps (specifically, at the middle area of the rectus femoris) located in the front of the thigh to disrupt microbubbles within the microcirculation (Figure 1B). SKM rectus femoris biopsies (500 mg) were taken from the right and left thigh of each animal. All samples were immediately processed after biopsy, placed in weigh boats and delivered to the sample processing room. These are procedures already established in our Lab (27). SKM (rectus femoris) was harvested (Figure 1A) for IHC from both the treated (left) and the control (right) thigh from the upper, middle and lower third of the muscle.

Figure 1A.

Non-invasive, site-specific UTMD gene therapy administered at middle region of rectus femoris. Yellow boxes show anatomical areas from where quadriceps (rectus femoris) was harvested. B. Delivery of genes by intravenously injecting plasmid DNA encapsulated in microbubbles into the animal’s blood stream through UTMD gene therapy. C. Diagrammatic representation of the constructs mixed with liposomes to form liposome-nucleic acid complexes. The composition is a DNA transposon-based vector. D. Direct detection of red fluorescent protein (DsRed) signal in baboon SKM. Strong signal for activity detected in the targeted side (right panel). No signal detected in the muscle-control. E. Antibody staining: Anti-Myf-5, a marker of SKM cells. In the right panel (bottom) the Myf-5 signal co-localized with Ds-Red. F. UTMD RT-PCR for DsRed mRNA only detected in SKM, and absent in heart, white adipose tissue (WAT), liver or spleen, confirming that the delivery was tissue-specific.

Delivery of plasmid cDNA constructs encoding the gene cocktail with BMP7/PRDM16/PGC-1a.

Plasmid-containing lipid-stabilized microbubbles were prepared as previously described (28). Briefly, a stock solution is prepared containing 270 mg of 1,2-dipalmitoyl- Sn-glycero −3- phosphatidylcholine, (Sigma, St. Louis, MO), 30 mg of 1,2- dipalmitoyl- Sn-glycero-3- phosphatidylethanolamine, (Sigma, St. Louis, MO), and 1 g of glucose. Plasmid containing microbubbles are prepared by mixing 2 mg of dried plasmid with 50 ul of lipofectamine 2000 (Invitrogen, Carlsbad, CA) and incubating at room temperature for 15 minutes. This liposome/plasmid DNA mixture is added to 250 ml of lipid stock solution, 50 ml of pure glycerol, and 5 ul of 10% albumin solution, mixed well with a pipette, and then placed in ice. Aliquots of 0.5 ml of this phospholipid-plasmid solution are placed in 1.5 ml clear vials; the air in the headspace of the vials is replaced with perfluoropropane gas (Air Products, Inc, Allentown, PA). The microbubbles appear as a milky white suspension floating on the top of a layer of liquid containing unattached plasmid DNA. The mean diameter and concentration of the microbubbles in the upper layer are measured by a particle counter (Beckman Coulter Multisizer III) (29).

Microbubble administration and ultrasound destruction.

Baboons received a functional cocktail 3-gene construct under the regulation of promoters incorporated into a phospholipid shell of perfluoropropane gas-filled microbubbles (29). The plasmid digestion, subcloning, ligation, isolation, and purification was performed by standard procedures and sequenced to confirm that no artifactual mutations were present (30). The microbubbles were infused intravenously. Plasmid DNA in microbubble suspension was infused at a constant rate for 10 min via pump (Genie, Kent Scientific, Torrington, CT, USA). During infusion, ultrasound was directed to the skeletal muscle (SKM) of the thigh (rectus femoris) to disrupt microbubbles within the microcirculation, using a commercially available ultrasound transducer (S3 probe, Sonos 5500, Philips Ultrasound, Bothell, WA, USA). Four bursts of ultrasound were triggered to every fourth end-systole by electrocardiogram using a delay of 45–70 ms after the peak of the R wave. This burst episodically destroyed the microbubbles in the SKM rectus femoris (29).

Body temperature monitoring using subcutaneously implanted data loggers.

To evaluate the stability and reliability of core body temperature for continuous body temperature monitoring, we collected long term temperature profiles from our pilot baboon by surgically implanting miniature temperature sensitive Star-Oddi’s small, leadless, implantable data loggers for temperature measurement subcutaneously in three different locations in the same animal (31). The 3 anatomic locations were 1) between the scapulae (shoulder blade) (SB), 2) in the control right thigh (RT) and 3) in the treated left thigh (LT). Temperature was measured daily throughout the entire experiment (Figure 2A).

Figure 2A.

Temperature measured with implanted subcutaneous data loggers. SB AGT vs. SB BGT P-value < 0.0001. LT AGT vs. LT BGT P-value < 0.0001. RT AGT vs. RT BGT P-value 0.0027. Two-tailed P-value: an alpha below 0.05, significant; over 0.05, not significant. (Mann Whitney test) Gene therapy administered on Sep-11–19 SB: Shoulder Blade. LT: Left Thigh. RT: Right Thigh. BGT: Before Gene Therapy. AGT: After Gene Therapy. B. Veterinary infrared thermography to assess brown adipose tissue (BAT) and UCP1 activity.

Veterinary infrared thermography (IRT).

Local temperature at both thighs through an infrared imaging technique (32) was measured. Surface temperature was recorded under anesthetized condition using an infrared digital thermographic camera (T660sc, emissivity of 0.98, FLiR Systems) placed 20 cm above the animal. The camera has a thermal sensitivity of ~0.1°C and a spatial resolution of 640 × 480 pixels (Figure 2B) (33).

Immunohistochemistry (IHC).

SKM tissue samples were fixed in 10% formalin for 24 hours and transferred into 70% alcohol for paraffin embedding and 4% paraformaldehyde and 20% sucrose overnight at 4 °C for frozen sections. The primary antibodies to evaluate overexpression in SKM for UCP1, and presence of PRDM16, PGC1a and BMP7, were added and incubated at 4°C overnight. After washing with PBS three times for 5 min each, the secondary antibody was added and incubated for 1 hour at room temperature Dapi (1:5,000 dilutions) staining for 5 min. Sections were rinsed three times with PBS for 5 min each and then mounted (10, 29).

Veterinary Care and Management.

Routine animal acclimation to technician presence was undertaken prior to the start dates of the procedures. Veterinary staff looked after the animals regarding food intake, attitudes (submissive, aggressive, etc.) and comfort level with presence of technical staff in bay. Another animal always accompanied the selected baboon in the bay (bay buddy). All animals were fasted 8–12 hours before anesthesia and procedures. Preanesthetic therapy was facilitated by injecting ketamine hydrochloride. Inhalation anesthesia was best achieved following endotracheal intubation. The endotracheal tube and intravenous catheter were removed as soon as the animal regained reflexes. The animals recovered in a well ventilated environment, and careful observation at frequent intervals was performed to minimize post-procedure complications. Routine analgesics were utilized when necessary.

Statistical analyses.

Repeated measures and two-way ANOVA were used to determine mean differences between and within group differences in experimental and control groups. Statistical analyses were carried out using SPSS (SPSS, Chicago, IL) and/or R (www.r-project.org).

Results

a). Individualized indirect calorimetry measurement.

We compared the REE (Kcal/d) measure with the indirect calorimeter with the Schoeninger mathematical equation used to predict REE from whole body mass in nonhuman primates (REE (Kcal/d) = 25.45 X body mass (Kg) + 172 (34). Figure 4 shows the mean REE for the male and female baboons (806 and 648 Kcal/d) and the mean REE from the equation (949.5 and 697 Kcal/d). Figure 4 also shows veterinary technicians measuring REE in two baboons.

Figure 4.

Establishing indirect calorimetry methods to measure REE, VO2, VCO2 and QT through a mobile indirect calorimeter in baboons. (Baboons 28114 and 26080 highlighted in yellow were first used as proof-of-concept to standardize the precise dosage of telazol and ketamine to achieve deep breathing relaxation and accurate REE measurements).

b). Targeting SKM by UTMD Gene Therapy in baboons.

We administered a DsRed reporter construct into the skeletal muscle of two baboons. Intravenous microbubbles carrying the reporter gene cocktail were destroyed within the microcirculation of muscle by ultrasound, achieving local gene expression after targeting our specific tissue. We were able to clearly demonstrate the ability to disrupt microbubbles and release the marker (DsRed). We used the right muscle of the thigh as the treated site, collecting tissues from the left muscle of the thigh in the same baboon to serve as the non-UTMD treated control site and performed confocal microscopy with FITC-labeled anti-MYF-5 with DsRed-labeled for immunohistology. A strong signal was observed (Figure 1 D, E) indicating detection of the DsRed reporter introduced into the muscle of the thigh by UTMD. These findings were confirmed by RT-PCR (Figure 1F).

c). BAT UCP1 overexpression in baboon SKM through UTMD gene therapy.

We administered plasmid cDNA constructs encoding BMP7/PRDM16/PGC-1a incorporated within microbubbles and intravenously delivered into the left thigh of one baboon. The right thigh of the same baboon was used as a control. An ultrasound transducer was directed to the baboon quadriceps (specifically, at the middle area of the rectus femoris) located in the front of the thigh to disrupt microbubbles within the microcirculation (Figure 1B). SKM (rectus femoris) was harvested (Figure 1A) for IHC from both the treated (left) and the control (right) thigh from the upper, middle and lower third of the muscle. Our IHC results showed a reliable pattern of effective UTMD-based gene delivery in enhancing SKM overexpression of the UCP1 gene in the middle area of the harvested muscle where we administered tissue-specific UTMD (Figures 5a and 5b). We observed a robust cellular presence of UCP1 in this anatomical topography (Figure 5a E and 5b A and B).

Figure 5a.

Robust UCP1 overexpression after UTMD gene therapy in left thigh (5aE) not found in the control right thigh (5aB). 5b. Rectus femoris middle area (left thigh) with abundant overexpression of UCP1 (5bA and B).

d). Changes in food consumption, body composition and weight.

The baboon received 1,600 Kcal/day of regular monkey chow before and after the administration of gene therapy. Daily average consumption for ~ 40 days was 878.7 Kcal/day with an average weight of 18.9 Kg before the gene therapy administration. After the administration of the UTMD gene therapy (~ 30 days), the average weight was maintained at 19.2 Kg. However, average food intake increased to 1453.9 Kcal/day. (Figure 6). Body composition measured by DXA reported body fat of 18.6% before the UTMD gene therapy and 18.3% after gene therapy administration, contrary to the lean body mass reporting an increase of almost 700 g (14.3 vs 15.0 Kg) (Figure 3).

Figure 6.

Mean energy intake before (878.7 cal.) and after (1453.9 cal.) UTMD-gene therapy. Steady weight was maintained during this period. Gene therapy was administered on 9/11/2019.

e). Data logger results.

The mean temperature between the shoulder blades (SB) (36 vs. 37 °C) and left thigh (LT) (36.4 vs. 38.2 °C) was significantly higher after gene therapy administration (Figure 2A). This was the same trend for the right thigh (RT), although the elevation was non-significant. The daily measurement curves show higher temperatures after gene therapy in all implanted sites.

f). IRT data.

We acquired images of the baboon whole body as well as the left and right thigh with our thermal imaging camera (FLIR). The temperature detected with IRT was higher after the UTMD gene therapy administration (Figure 2B). Measurements showed 92.5 °F before gene therapy vs. 93.0 °F after gene therapy in whole body; 93.1 °F before gene therapy vs. 95.4°F after gene therapy in the left thigh; and 93.3°F before gene therapy vs 93.8°F after gene therapy in the right thigh.

Discussion

We have developed novel gene therapeutic strategies in baboons using state-of-the-art technologies including resting energy expenditure (REE) measured with the indirect calorimeter Breezing, UTMD, body temperature monitoring using subcutaneously implanted data loggers, and infrared imaging. NHP models, including the baboon, are essential to bridge the translational research gap between preclinical animal and human clinical research trials (28, 35).

There have been a few attempts to utilize indirect calorimetry in NHP to study changes of energy expenditure in relation to energy intake (36–40). None of these studies utilized an indirect calorimetry system that obtained continuous measurements of both oxygen and carbon dioxide concentrations while maintaining the comfort of the non-human primates. As shown in Figure 4, by establishing a unique method to measure REE, VO2, VCO2 and QT through the Breezing® mobile indirect calorimeter in baboons, we will be able to obtain accurate and valuable data regarding thermogenesis and REE before and after UTMD gene therapy and for many other cardiometablic research studies. To the best of our knowledge, this is the first report describing the use of a personalized and portable indirect calorimeter in captive baboons.

There is strong interest in the possibility of increasing depots of brown adipose tissue (BAT) as a way to reduce excess fat and improve metabolic homeostasis. Our study adapted rodent protocols for non-viral gene therapy to study fat tissue biology and immunometabolism in the baboon (41). Non-viral ultrasound-targeted microbubble destruction (UTMD) has emerged as a significant means of non-invasive, tissue-specific gene therapy (17). We have previously employed a tissue specific approach to deliver a gene cocktail (cyclin D2, CDK4, and GLP-1) (29) incorporated in microbubbles into the pancreas of baboons that had previously undergone beta-cell destruction after streptozotocin (STZ) administration (42). Gene therapy by UTMD achieved in vivo normalization of the intravenous glucose tolerance test curves in STZ hyperglycemic-induced conscious tethered baboons (29).

We recently demonstrated that the delivery of a PRDM16/PGC-1α/BMP7 gene cocktail to skeletal muscle (SKM) in mice and rats using gene therapy (9), induced a BAT phenotype, increasing thermogenesis and weight loss in these rodents (10). Our group has recently delivered plasmid cDNA constructs to baboon’s thigh SKM encoding the same gene cocktail with BMP7/PRDM16/PGC-1a using UTMD gene therapy. We were able to induce overexpression of uncoupling protein-1 (UCP1), a marker for brown adipose tissue (BAT) and differentiate myogenic precursors to fully differentiated brown fat adipocytes as it is shown Figures 1A, B and C, 5a and 5b. The right thigh of the same baboon was used as a control. Data from Figures 5a and 5b clearly indicated that our plasmid DNA construct encoding the gene combination of PRDM16, PGC-1a and BMP7 reprogrammed adult SKM into brown adipose cells in vivo. IHC showed that a UCP1 signal was detected in baboon SKM when the UTMD-PRDM16/BMP7/PGC-1α gene cocktail was delivered through UTMD technology to the site-specific left thigh (Figures 5a E, 5b A and B), but was not seen in the right thigh control (upper, middle, lower area of the right rectus femoris) (Figure 5a A, B and C). We therefore established proof-of-concept that site-specific administration of BMP7/PRDM16/PGC-1a to SKM utilizing UTMD gene therapy engineered a BAT phenotype with UCP1 over-expression (Figure 1C). The cocktail of three thermogenic brown adipose tissue genes (PRDM16, BMP7, PGC-1α) are delivered into SKM through the UTMD method (10). To the best of found knowledge, the combination of tissue-specific gene therapy administered to SKM of baboons to engineer BAT, transposon-based vectors and microbubbles (UTMD) is unique in NHP research.

White adipose tissue (WAT) and BAT have distinct developmental origins (15). Unlike WAT that originate from Myf5 negative (–) precursors and are derived from blood vessel-associated pericyte-like cells, SKM and BAT are both derived from precursors expressing the key myogenic factor Myf5 positive (+) (43). From an evolutionary perspective, the ability of BAT to produce facultative thermogenesis became so important that its contractile components disappeared. Unlike WAT large amounts of mitochondria appeared, and a thermogenic tissue of myogenic origin developed with a strong expression of the nuclear gene UCP1, the uncoupler of oxidative phosphorylation responsible for non-shivering thermogenesis (44). Hence, some authors currently consider BAT as specialized muscle (9, 45). Because SKM and BAT derive from Myf5+ cells and SKM has abundant precursors to allow facilitate healing/regeneration, SKM is an ideal target for BAT rather than WAT. SKM also has better vascularization than WAT to allow and facilitate healing/regeneration. Indeed, at rest, SKM muscle blood flows may reach 4 ml/min per 100g (46); maximal blood flows may reach 50–100 ml/min per 100g depending upon the muscle type. Therefore, blood flow can increase 20 to 50-fold. Adipose tissue blood flow measured in the overnight-fasted state is typically close to 2 ml/min per 100 g tissue (47). Although it is close to values measured in resting skeletal muscle, inert fat stores occupy most of the volume of adipose tissue. Due to our non-viral tissue-specific UTMD-gene therapy method, where the delivery of the microbubbles needs the presence of an abundant blood flow, SKM is a better tissue to target for BAT. The single plasmid gene dosage administered in this feasibility study was 12 mg of plasmids calculated for an 18.5 Kg baboon (0.6 mg/Kg). We have previously delivered 1 mg of plasmids/kg of baboon weight. We propose to use 1 – 1.5 mg of plasmids x Kg of body weight in our future studies to optimize efficacy and screen for safety and adverse effects.

The microscopic ultrastructure of the harvested SKM tissue illustrates the effects of ultrasound-mediated microbubble destruction (UTMD) efficacy (Figures 5a and 5b). This includes radiation pressure generated by ultrasound transmission through the SKM that can promote the local accumulation of blood cells by pushing them to the vascular wall (48). The ultrasound mechanical and sonic cavitation effects due to its wave motion induces contraction of endothelial cells, subsequent destruction of micro-vessels and, most importantly, the widening of gaps between cells (49). There is also a shear stress generated by microbubble rupture that can help the in vivo plasmid-based gene transfer in the blood vessel to penetrate the vessel wall and enter into skeletal muscle tissue. It has been reported that UTMD can obliterate the SKM blood capillaries, inducing local infiltration of erythrocytes and promoting cellular penetration of the endothelial physiologic barriers (50). Low-frequency ultrasound associated with microbubble destruction also increases the efficiency of gene transfer into skeletal muscle (51). In addition, a slight aseptic inflammatory response induced by microvascular fracture during ultrasound-mediated microbubble destruction can promote neovascularization which may increase blood flow in the skeletal muscle (52). This neovascularization is critical for the appearance and growth of new brown adipocytes from myf5+ cells committing to brown preadipocytes. All these biological effects seem to have occurred in the intervened left SKM of our baboon after UTMD. Indeed, the observation of the microscopic ultrastructure of the cells in Figure 5a D, E and F and Figure 5b clearly shows that the integrity of the cells was disrupted. Widened gaps within the cells were seen in this ultrasound microbubble destruction cellular group (left SKM), but not in the cell control group from the right SKM that did not get exposure to UTMD (Figure 5a A, B and C).

We also observed interesting short-term changes in daily temperature monitored with data loggers, metabolic heat measured with veterinary IRT imaging, as well as in key metabolic parameters such as SKM insulin-mediated glucose disposal, and food consumption and fat mass evaluation with DXA before and after UTMD gene therapy in our intervened animal. As shown in the results, the mean temperature between the SB, LT, RT and daily temperature measurement curves was discretely higher after gene therapy administration (Figure 2A). This thermogenesis after UTMD gene therapy may have contributed to the steady weight after gene therapy in spite of an increase in calorie intake (Figure 6). Due to the thermogenic properties of BAT, thermal imaging is a potential method for detecting BAT and studying its function. Infrared thermography (IRT) is an established and accepted technique for studying BAT function in animals (53). Studies employing IRT to assess BAT activities in humans have recently been published (33). We assessed BAT and UCP1 activity with IRT. As with the thermologger measurements, the temperature detected with IRT was higher after the UTMD gene therapy administration (Figure 2B). However, the measurements were performed only one week after UTMD administration.

The changes in food consumption, body composition and weight deserve greater attention. The baboon received 1,600 Kcal/day of regular monkey chow before and after the administration of gene therapy. Daily average consumption for ~ 40 days was 878.7 Kcal/day with an average weight of 18.9 Kg before the gene therapy administration. After the administration of the UTMD gene therapy (~30 days), the average weight was maintained at 19.2 Kg. However, average food intake increased to 1453.9 Kcal/day. The animal did not gain weight despite an almost doubling of calorie intake (Figure 6). Interestingly, body composition measured by DXA reported body fat of 18.6% before the UTMD gene therapy and 18.3% after gene therapy administration, contrary to the lean body mass reporting an increase of almost 700 g (14.3 vs 15.0 Kg) (Figure 3B). Overall, these results could explain a trend for increased energy expenditure to compensate for the increase in calorie intake, reflected by a steady body weight before and after the UTMD gene-based therapy that could be the result of an overexpression of UCP1 in SKM and thermogenesis. This was the same observation in food intake and weight found in our previous proof of concept for UTMB gene delivery into the left thigh of obese Zucker rats (9).

We replicated our Zucker rat study in Sprague-Dawley (SD) rats to test our hypothesis in a diet-induced obesity wild type rat. UCP1 signal was detected in rat SKM cells one month after UTMD-PRDM16/BMP7/PGC-1α was delivered (54). The highest temperature elevation measured by infrared thermography was in the treated left thigh after delivering the UTMD gene cocktail. Following a similar food intake pattern as the one observed in the Zucker rats, weight loss in SD rats treated with UTMD did not recover to the levels of controls in spite of food intake recovery. These findings from Zucker rats (9) and SD rats (54) show that we can achieve an energy gap with the UTMD-PRDM16/PGC-1a (55) that perhaps explains why treated rats gradually recovered their food intake patterns while never recovering weight to control animal levels. Similar to our baboon, energy expenditure represented by thermogenesis likely explains the weight loss maintained in these rats regardless of the gradual recuperation of food intake, clearly indicating BAT UCP1 over activity and metabolic heat production secondary to UTMD gene therapy in SKM and UCP1 overexpression. These findings follow, to a certain extent, similar explanations for the efficacy of long-term weight loss achieved by bariatric surgery and its effectiveness to stimulate daily energy expenditure as the energy gap (56) for long-term maintenance without apparent extreme changes in daily energy intake. In animal models, post-surgery increased bile acids (BA) might also impact energy expenditure. Binding of BA TGR5 receptors in SKM and BAT may contribute to an enhanced action of thyroid hormones by increasing energy expenditure (57).

In summary, methods including resting energy expenditure (REE) measured with the indirect calorimeter Breezing, UTMD, body temperature monitoring using subcutaneously implanted data loggers, and veterinary IRT imaging were used to characterize the molecular mechanism that regulates food intake, adipose tissue biology, energy expenditure, fatty-acid oxidation and insulin-glucose axis regulation. These research resources allow the engineering of SKM cells into a BAT phenotype through UTMD technology (Figure 7), inducing overexpression of UCP1 to achieve an increase in REE and thermogenesis, and facilitating immunometabolic improvements. These innovative methods are poised to significantly improve basic and translational research in energetics, physiology, and health.

Figure 7.

Research methodology development to engineer SKM cells into a BAT phenotype through UTMD technology.

Highlights.

• Baboons are an outstanding NHP model for immunometabolic research.

• Innovative technologies and advanced research strategies for energetics and translational medicine with this NHP model are reported.

• Methods developed to measure REE (indirect calorimeter), temperature (thermologgers and infrared imaging), and glucose uptake (euglycemic clamps).

• We engineered SKM cells into a BAT phenotype through UTMD technology in baboons.