Abstract
Background
Metabolic dysfunction-associated steatotic liver disease (MASLD) is increasingly recognized in people with HIV (PWH), with both HIV and antiretroviral therapy contributing to liver damage and glucose intolerance. However, the role of viral proteins derived from reservoirs in this process remains unclear.
Methods
Adeno-associated virus (AAV) constructs encoding a control protein or HIV-1 viral protein R (Vpr) driven by the thyroxine-binding globulin promoter were administered to male mice (n = 5 per group) fed regular chow or a high-fat diet (HFD). Young adult mice underwent intraperitoneal glucose tolerance testing and magnetic resonance imaging, followed by euthanasia. Liver and adipose tissues were analyzed for mRNA expression, lipid levels, and fat content and plasma samples for triglycerides and liver function.
Results
AAV-Vpr mice on HFD developed exacerbated hepatic steatosis, glucose intolerance, and systemic inflammation compared to AAV-green fluorescent protein control mice. Gene expression indicated enhanced de novo lipogenesis, diminished lipid oxidation and insulin resistance in the liver. These effects were distinct from those observed with HFD alone, confirming a Vpr-specific contribution.
Conclusion
Vpr upregulates the hepatic synthesis of fatty acids and downregulates their oxidation and export as triglycerides. The liver-specific activity of Vpr is sufficient, in synergy with a HFD, to cause hepatic steatosis and impaired glucose tolerance. These findings define a tissue-autonomous role for Vpr in mediating hepatic steatosis in mice, with implications for MASLD development and its complications in PWH.
Keywords: metabolic dysfunction-associated steatotic liver disease, MASLD, Vpr, metabolism, mice, adipose tissue
HIV and antiretroviral therapy (ART) are associated with significant metabolic alterations, including adipose tissue (AT) dysfunction, hepatic steatosis, dyslipidemia, and insulin resistance. These changes contribute to the development of metabolic dysfunction-associated steatotic liver disease (MASLD) in people with HIV (PWH), which has become the most prevalent liver disease worldwide [1, 2]. Among PWH, MASLD is the leading cause of chronic liver disease [3], with a prevalence exceeding 50% in some cohorts [4-10] and a higher prevalence in males, particularly those with obesity, type 2 diabetes, or metabolic syndrome [11-13]. Given the high prevalence of metabolic complications in PWH, understanding HIV-specific contributors to MASLD in this population is critical for improving patient outcomes.
HIV-1 may contribute directly to MASLD through several mechanisms, including acceleration of de novo lipogenesis, inhibition of fatty acid β-oxidation, and impaired triglyceride (TG) export [14] from the liver. Additionally, HIV-associated chronic inflammation and immune activation can worsen metabolic dysfunction, leading to liver damage and fibrosis. PWH have greater severity of MASLD than individuals without HIV, as evidenced by greater severity of metabolic dysfunction-associated steatohepatitis and a higher rate of progression to metabolic dysfunction-associated steatohepatitis (63% compared to 37% in persons without HIV) [15].
In prior work, we and others demonstrated that the HIV-1 accessory protein viral protein R (Vpr) plays a critical role in driving HIV-specific metabolic abnormalities [14, 16-22]. Vpr circulates in the serum of PWH even when on suppressive ART and has been shown to significantly and independently affect glucose and lipid metabolism and AT function [17]. Using 2 distinct mouse models—a transgenic model, in which Vpr is expressed under the control of the PEPCK promoter and secreted into the circulation, and a pharmacologic model, in which synthetic Vpr is continuously infused into mice—our prior work revealed that Vpr-induced metabolic dysfunction recapitulates several of the characteristic defects observed in PWH, namely insulin resistance, dyslipidemia, abnormal AT thermogenesis, and fatty liver disease [14, 16-20].
The animal models and design of the previous studies did not permit us to determine whether fatty liver disease is caused by Vpr's actions specifically in the liver or by a combination of multiorgan or systemic effects, including AT dysfunction and whole-body insulin resistance and inflammation. Hence, in the present study, we asked whether Vpr expressed only in mouse liver is sufficient to cause hepatic steatosis. To answer this question, we utilized adeno-associated virus (AAV) delivery to express Vpr under the control of the thyroxine-binding globulin (TBG) promoter, to ensure liver-specific expression of Vpr. We hypothesized that this model, when combined with a high-fat diet (HFD; a necessary substrate), would confirm the Vpr-induced hepatic steatosis phenotype and help elucidate the mechanism of hepatic steatosis development in PWH.
Materials and Methods
Animal Study
A total of 25 male C57BL/6J mice were used. Mice were randomly assigned to 5 experimental groups (n = 5 per group): (1) regular diet + AAV-green fluorescent protein (GFP) (control vector), (2) regular diet + AAV-Vpr, (3) HFD+ AAV-GFP (control vector), (4) HFD + PBS (nonvector control; data not shown), and (5) HFD + AAV-Vpr. Experimental conditions and sample sizes were based on previous studies investigating the effects of Vpr on AT [14, 17].
AAV Mouse Model
Construction
A self-complementary AAV transfer vector with TBG (liver-specific) promoter was used to clone Vpr or GFP (control). AAV was packaged by triple transient transfection (AAV Transfer Shuttle, AAV8 Rep/Cap serotype, AdDF6 helper plasmid) into 293T cells using iMFectin transfection reagent (GenDEPOT, Houston, TX, USA). A total of 60 × 15-cm dishes were transfected; cell-associated and media-secreted AAV were collected separately 3 days after transfection. Cell-associated AAV was recovered by cell lysis, and media-secreted AAV was precipitated by polyethylene glycol. These were combined and purified over an iodixanol density gradient. AAV titer was quantified by absolute quantitative PCR with the primers corresponding to TBG.
Administration
AAV constructs were used to express Vpr under the control of the TBG promoter or, as controls, AAV-expressing GFP. AAV-Vpr or AAV-GFP or saline was introduced into 7- to 8-week-old male C57BL/6J mice via tail vein injection. The animals were divided into 2 dietary groups: (1) regular chow or (2) HFD. The regular chow diet provided 13% kcal from fat, 62% from carbohydrates, and 25% from protein. The HFD provided 60% kcal from fat, 20% from carbohydrates, and 20% from protein. Body weight and food intake were measured weekly starting at week 8.
Experimental Timeline
Regular chow group
Mice received regular chow for all 15 weeks. AAV-GFP (vector control) or AAV-Vpr or saline (nonvector control) was given via tail vein injection at the age of 7 to 8 weeks. Intraperitoneal glucose tolerance test (IP-GTT) and magnetic resonance imaging (MRI) were done at 13 weeks of age and euthanasia at 15 weeks (Fig. 1A).
Figure 1.
Schematic diagram showing experimental timeline for (A) regular chow: mice received regular chow for all 15 weeks. AAV-GFP or AAV-Vpr was given via tail vein injection at the age of 7 to 8 weeks. IP-GTT and MRI were done at 13 weeks of age, and animals were fasted overnight (14 to 16 hours) followed by euthanasia at 15 weeks. (B) HFD groups: mice received regular chow for the first 7 weeks of life. AAV-GFP or AAV-Vpr was given via tail vein injection at the age of 7 to 8 weeks. HFD was started at the age of 7 to 8 weeks at the time of AAV administration. HFD mice underwent IP-GTT and MRI at 21 weeks, with energy balance studies performed at 23 weeks, and animals were fasted overnight (14 to 16 hours) followed by euthanasia at 25 weeks.
Abbreviations: AAV, adeno-associated virus; GFP, green fluorescent protein; HFD, high-fat diet; IP-GTT, intraperitoneal glucose tolerance test; MRI, magnetic resonance imaging; Vpr, viral protein R.
HFD group
Mice received regular chow for the first 7 weeks of life. AAV-GFP (vector control) or AAV-Vpr or saline (nonvector control) was given via tail vein injection at the age of 7 to 8 weeks. HFD was started at 7 to 8 weeks at the time of AAV administration. HFD mice underwent IP-GTT and MRI at 13 and 21 weeks, with energy balance studies performed at 23 weeks and euthanasia at 25 weeks (Fig. 1B). Animals were fasted overnight (14 to 16 hours). Animals were anesthetized using isoflurane (2-3% oxygen) for terminal procedures. Euthanasia was performed under deep anesthesia, by cervical dislocation, in accordance with Institutional Animal Care and Use Committee-approved protocols.
At the time of euthanasia, blood was collected via retro-orbital bleed. Approximately 100 to 200 μL of blood was collected into heparin-coated tubes. Samples were centrifuged at 2000 × g for 5 minutes at 4 °C to obtain plasma. Plasma was stored at −80 °C. Tissues were flash-frozen in liquid nitrogen for protein expression and mRNA analyses. Liver tissues were also embedded in optimal cutting temperature compound for Oil Red O staining or fixed in 10% formalin for histology.
Body composition analysis by MRI
For measurement of whole-body fat mass, lean mass, and free fluid and total body water, the nuclear magnetic resonance system EchoMRI-900™ (Echo Medical System, Houston, TX, USA) was used without the need for anesthesia, per the manufacturer's instructions.
IP-GTT
Mice were fasted for 16 hours before intraperitoneal glucose administration (1.5 g/kg body weight). Blood glucose measurements were taken using a handheld glucometer at baseline (0 minutes) and at 15, 30, 60, and 120 minutes post-glucose administration.
Hepatic TG content
Liver tissue (∼100 mg) stored at −80 °C was homogenized in phosphate-buffered saline, and protein concentration was quantified using the DC Protein Assay Kit (Bio-Rad, Hercules, CA, USA). Lipids were extracted using chloroform: methanol (2:1) with 0.1% sulfuric acid. TG content was determined using commercially available kits (Wako Chemicals, Richmond, VA, USA) in microtiter plates and normalized to the protein concentration of the homogenate.
Histology and Oil Red O staining
Embedded liver was cryo-sectioned (4 µm), then cut and stained with hematoxylin-eosin for general histology or Oil Red O for fat content. The sections were analyzed using a Keyence microscope (Keyence Corporation, Itasca, IL, USA) at 10 to 20× magnification, and fat content was quantified using ImageJ software.
Energy balance studies
Energy expenditure, activity levels, and food/water intake were measured using a Comprehensive Laboratory Animal Monitoring System-Home Cage system (Columbus Instruments, Columbus, OH, USA). Indirect calorimetry data were analyzed using CalR software [23], which assessed the statistical significance of energy balance differences between groups.
Plasma metabolite measurement
Fasting (14-16 hours) plasma levels of TG (Thermo Fisher Scientific, Grand Island, NY, USA), glucose (Thermo Fisher Scientific), nonesterified fatty acids (Wako Chemicals, Richmond, VA, USA), and total cholesterol (Abcam, Waltham, MA, USA) were measured using standard commercial assay kits.
mRNA expression analysis
Total RNA was extracted from liver tissues using Trizol (Invitrogen, Carlsbad, CA, USA). The RNA concentration and integrity were assessed using a NanoDrop spectrophotometer and agarose gel electrophoresis, respectively, and cDNA was synthesized using the RNA-to-cDNA kit (Applied Biosystems, Grand Island, NY, USA). Real-time PCR was performed using the TaqMan assay (Applied Biosystems) to measure Vpr gene expression and target genes for gluconeogenesis (Pepck: assayID: Mm01247058_m1, Pcx: assay ID: Mm00500992_m1, and G6Pc: assayID: Mm00839363_m1), de novo lipogenesis (Srebp1c: assay ID: Mm00550338_m1, Chrebp: assay ID: Mm02342723_m1, Lpk: assay ID: Mm00443090_m1, Dgat: assay ID: Mm00515643_m1, Fasn: assay ID: Mm00662319_m1, Scd1: assay ID: Mm00772290_m1, and Acc: assay ID: Mm01304273_m1), β-oxidation (PPARα: assay ID: Mm00440939_m1, Cpt1α: assay ID: Mm00550438_m1, Aox: assay ID: Mm00443579_m1, Ehhadh: assay ID: Mm00619685 _m1, Acaa2: assay ID: Mm00624282_m1, and Lcad: assay ID: Mm00599660_m1), very low density lipoprotein (VLDL)-TG export (Mttp: assay ID: Mm00435015_m1), and inflammatory genes (Il6: assay ID: Mm00446190_m1, Il10: assay ID: Mm01288386_m1, and TNFa: assay ID: Mm00443258_m1) were quantified with a ABI 7000 Real-Time PCR System (Applied Biosystems). Cycling conditions were 95 °C for 10 minutes, then 50 cycles of 95 °C for 15 seconds (s), 60 °C for 30 seconds, and 72 °C for 30 seconds. Pgk1 was used as the housekeeping gene for normalization. Relative mRNA expression was calculated using the 2−ΔΔCt method normalized to Pgk1.
Animal study approval
All animal protocols were approved by the Baylor College of Medicine Institutional Animal Care and Use Committee. The study adhered to the guidelines outlined in the “Guide for the Care and Use of Laboratory Animals” (National Institutes of Health publication 86-23, revised 1985). Mice were housed in the Baylor College of Medicine Taub's animal care facility and received humane care [14, 17].
Statistical analysis
Data are expressed as the mean ± SE. Statistical analyses were performed using Mann-Whitney, 2-tailed, unpaired t-tests for unequal variance to compare the 2 groups. A type I error rate (P) < .05 was considered statistically significant. Graphical representation and statistical analyses were carried out using GraphPad Prism software (GraphPad Software, Boston, MA, USA).
Results
Body Composition and Liver Weight in AAV-Vpr Male Mice
We first checked Vpr expression in animals receiving AAV-TBG-Vpr compared to AAV-GFP: Mice receiving TBG-Vpr had increased Vpr expression compared to mice expressing AAV-GFP (Fig. 2A and 2F). We then assessed the effects of Vpr expression on body composition in AAV-Vpr and control AAV-GFP mice at 13 (regular chow) and 21 weeks (HFD) of age. On regular chow, there were no significant differences in lean mass, fat mass, total body weight, intra-abdominal fat weight, perigonadal fat, weight, brown AT weight, or liver weight between the 2 groups (Fig. 2B–2E). At 25 weeks of age (following 18 weeks of HFD), AAV-Vpr mice had significantly greater liver weights compared to AAV-GFP mice (4.1 ± 0.25 vs 3.2 ± 0.26 g, P = .008), but overall lean mass; fat pad weight; total body weight; and compartmental weights of intra-abdominal fat, perigonadal fat, retroperitoneal fat, and brown AT showed no between-group differences (Fig. 2G-2J).
Figure 2.
Liver weight of AAV-Vpr mice is increased compared to AAV-GFP mice after 25 weeks of a high-fat diet. (A) Vpr expression in AAV-Vpr mice compared to AAV-GFP mice on regular chow. (B-E). The body composition of the mice on regular chow diet at the ages indicated: (B) lean body mass; (C) fat mass; (D) total body weight; (E). weights of adipose tissue compartments and liver, as percentage of body weight. (F) Vpr expression in AAV-Vpr mice compared to AAV-GFP mice on a high-fat diet. (G-J) Body composition of the mice on a high-fat diet at the ages indicated: (G) lean body mass; (H) fat mass; (I) total body weight; (J) weights of adipose tissue compartments and liver, as percentage of body weight. n = 5 mice in each group. Values mean ± SE. Two-tailed, unpaired t-tests for unequal variance were used. P < .05 was considered significant. **P < .01.
Abbreviations: AAV, adeno-associated virus; GFP, green fluorescent protein; IF, intra-abdominal fat; PGF, perigonadal fat; RPF, retroperitoneal fat; Vpr, viral protein R.
Energy Balance in AAV-Vpr Mice
Oxygen consumption (Fig. 3A–3B), carbon dioxide production (Fig. 3C–3D), respiratory exchange ratio (Fig. 3E–3F), energy expenditure (Fig. 3G–3H), and cumulative food intake (Fig. 3I–3J) were similar between AAV-Vpr and AAV-GFP mice on HFD at 23 weeks of age. These results suggest that Vpr expression does not alter overall energy balance or physical activity when lean mass is a covariate.
Figure 3.
Energy balance is similar in AAV-Vpr mice and AAV-GFP mice on a high-fat diet at 24 weeks of age. (A-B) Oxygen consumption; (C-D) carbon dioxide production; (E-F) respiratory exchange ratio; (G-H) energy expenditure; (I-J) total food intake over 1 week in mice aged 13 weeks. n = 5 mice in each group. Values mean ± SE. Two-tailed, unpaired t-tests for unequal variance were used. P < .05 was considered significant.
Abbreviations: AAV, adeno-associated virus; GFP, green fluorescent protein; Vpr, viral protein R.
Glucose Intolerance in AAV-Vpr Mice
AAV-Vpr mice exhibited glucose intolerance compared to control AAV-GFP mice when fed both regular chow and HFD. On regular chow at 13 weeks of age, the areas under the curve calculated from glucose tolerance tests were significantly greater in AAV-Vpr mice compared to AAV-GFP mice (44 426 ± 2069 vs 37 004 ± 1129 mg.min/dL, P = .02) (Fig. 4A–4B). This difference was also observed at 23 weeks of age in AAV-Vpr vs AAV-GFP mice on HFD (39 890 ± 1542 vs 32 932 ± 1270 mg.min/dL, P = .02) (Fig. 4C–4D). These data indicate that isolated hepatic Vpr expression leads to glucose intolerance independent of diet. Insulin levels in IP-GTT samples were not different in AAV-Vpr mice compared to AAV-GFP mice on HFD (Fig. 4E–4F). Mice on HFD that received AAV-Vpr compared to AAV-GFP displayed elevated levels of gluconeogenesis genes Pepck and Pcx (Fig. 4G), whereas G6pc was not different in the 2 groups (Fig. 4G).
Figure 4.
Glucose concentration is altered in AAV-Vpr and AAV-GFP mice on a high-fat diet as well as regular chow. (A) Altered glucose concentration curves in AAV-Vpr (red) and AAV-GFP (black) mice. (B) Bar graphs of AUCs of glucose. (C-D) Glycemic responses during IP-GTT of mice on a high-fat diet at 21 weeks of age with (C) glucose concentration curves in AAV-Vpr (red) and AAV-GFP (black) mice. (D) Bar graphs of AUC of glucose. (E) Insulin levels during IP-GTT in mice on a high-fat diet. (F) Bar graphs representing the AUC of insulin during IP-GTT in mice on a high-fat diet. (G) Elevated levels of gluconeogenesis genes (Pepck and Pcx) in AAV-TBG-Vpr mice compared to AAV-GFP mice. n = 5 in each group. Two-tailed, unpaired t-tests for unequal variance were used. P < .05 was considered significant. Values mean ± SE. *P < .05.
Abbreviations: AAV, adeno-associated virus; AUC, area under the curve; GFP, green fluorescent protein; IP-GTT, intraperitoneal glucose tolerance test; TBG, thyroxine-binding globulin; Vpr, viral protein R.
Increased Hepatic Fat Accumulation in AAV-Vpr Mice on HFD
On regular chow, AAV-Vpr mice had lower intrahepatic TG (IHTG) levels compared to AAV-GFP mice (13.27 ± 0.71 vs 23.72 ± 2.2 mg/g liver, P = .02) (Fig. 5A). However, following HFD, AAV-Vpr mice had significantly higher IHTG than AAV-GFP mice (63.0 ± 4.7 vs 40.26 ± 2.6 mg/g liver, P = .02) (Fig. 5B). Oil Red O staining of liver tissue revealed a 2.3-fold increase in neutral lipids in the AAV-Vpr mice on HFD compared to the AAV-GFP mice on HFD (24.89 ± 1.4% vs 10.69 ± 2.2% area, P = .02) (Fig. 5C–5D). However, no significant differences were observed in plasma TG (Fig. 5E), total cholesterol (Fig. 5F), glycerol (Fig. 5G), or nonesterified fatty acids concentrations (Fig. 5H) between AAV-Vpr and AAV-GFP mice on HFD, indicating that the liver-specific effects of Vpr on hepatic fat accumulation are not reflected in circulating lipid profiles.
Figure 5.
AAV-Vpr develops hepatic steatosis on high-fat diets. (A) Intrahepatic triglyceride levels in AAV-Vpr compared to AAV-GFP mice on regular chow. (B) Intrahepatic triglyceride levels in AAV-Vpr compared to AAV-GFP mice on high-fat diets. (C) Oil Red O and hematoxylin-eosin staining in liver sections of the mice on a high-fat diet (10× magnification). (D) Quantification of Oil Red O staining in liver sections by Image J software in AAV-GFP vs AAV-Vpr mice on a high-fat diet. (E-H) Plasma levels of triglycerides (E), total cholesterol (F), glycerol (G), and nonesterified fatty acids (H). n = 5 in each group. Two-tailed, unpaired t-tests for unequal variance were used. P < .05 was considered significant. Values mean ± SE. *P < .05, **P < .01.
Abbreviations: AAV, adeno-associated virus; GFP, green fluorescent protein; Vpr, viral protein R.
Elevated Levels of Inflammatory Genes in Adipose Tissue of AAV-Vpr Mice on HFD
To assess whether hepatic Vpr expression induces local or systemic inflammation, we performed quantitative PCR for key inflammatory genes (Il6, Il10, and TNFa) in the liver and perigonadal (visceral) fat of AAV-Vpr and AAV-GFP control mice. No significant differences were observed in the hepatic expression of these inflammatory markers between the 2 groups (Fig 6A), suggesting that liver-targeted Vpr does not induce a strong local inflammatory response. In contrast, perigonadal fat from AAV-Vpr mice showed significant upregulation of Il6 and Il10 (Fig 6B), indicating that hepatic Vpr expression may exert systemic effects on AT inflammation. These findings suggest liver Vpr expression causes broader tissue-extrinsic and inflammatory effects.
Figure 6.
Inflammatory gene expression in liver and adipose tissue of AAV-Vpr and AAV-GFP mice. (A) Expression of inflammatory markers (Il6, Il10, and Tnfα) in liver tissue of AAV-Vpr and AAV-GFP mice on a high-fat diet, assessed by quantitative PCR. No significant differences were observed. (B) Expression of the same markers in perigonadal adipose tissue revealed significant upregulation of Il6 and Il10 in AAV-Vpr mice compared to controls, indicating a systemic inflammatory effect linked to hepatic Vpr expression. n = 5 in each group. Two-tailed, unpaired t-tests for unequal variance were used. P < .05 was considered significant. Values mean ± SE. *P < .05, **P < .01.
Abbreviations: AAV, adeno-associated virus; GFP, green fluorescent protein; Vpr, viral protein R.
Gene Expression Changes in Lipogenesis, Fatty Acid Oxidation, and Lipid Transport in AAV-Vpr Mice on HFD
We next investigated molecular features underlying the increased hepatic fat accumulation in AAV-Vpr mice by measuring gene expression related to lipogenesis, fatty acid oxidation, and lipid transport. On regular chow, there were no significant differences in the expression of key genes involved in these pathways between AAV-Vpr and AAV-GFP mice (Fig 7A-7E). However, on HFD, AAV-Vpr mice exhibited significant alterations in mRNA expression of the following classes of genes: AAV-Vpr mice showed increased expression of genes regulating de novo lipogenesis, including Srebp1c (1.7-fold) and Chrebp (1.7-fold) and its target gene Lpk (2-fold) (Fig. 7F). The mRNA levels of Dgat (2.3-fold), Fasn (2-fold), Scd1 (21-fold), and Acc (1.7-fold) were also increased (Fig. 7G), indicating enhanced capacity for lipogenesis. In parallel, AAV-Vpr mice on HFD displayed downregulation of key fatty acid oxidation genes, including Pparα (0.2-fold), Cpt1α (0.26-fold), Aox (0.67-fold), Ehhadh (0.13-fold), Acaa2 (0.5-fold), and Lcad (0.55-fold) (Fig. 7H-6I), indicating reduced capacity for fatty acid oxidation. Expression of Mttp (the gene encoding microsomal TG transfer protein) was also decreased by 0.46-fold in AAV-Vpr mice on HFD (Fig. 7J), which could contribute to impaired VLDL-TG export from the liver. These gene expression data indicate that Vpr in the liver, when combined with the metabolic stress of HFD and obesity, dysregulates metabolic gene expression in a direction that enhances lipogenesis while inhibiting fatty acid oxidation and TG export, promoting hepatic fat accumulation.
Figure 7.
Altered gene expression in liver AAV-Vpr compared to AAV-GFP mice on a high-fat diet. (A) mRNA expression of Srebp1c, Chrebp, and Lpk is similar in AAV-Vpr and AAV-GFP mice on regular chow. (B) mRNA expression of Dgat, Fasn, Scd1, and Acc is similar in AAV-Vpr and AAV-GFP mice on regular chow. (C) mRNA expression of PPARa is similar in AAV-Vpr and AAV-GFP mice on regular chow. (D) mRNA expression of Cpt1, Aox, Ehhadh, Acc2, and Lcad is similar in AAV-Vpr and AAV-GFP mice on regular chow. (E) mRNA expression of MTP is similar in AAV-Vpr and AAV-GFP mice on regular chow. (F) Increased mRNA expression of Srebp1c, Chrebp, and Lpk in AAV-Vpr compared to AAV-GFP mice on a high-fat diet. (G) Increased mRNA expression of Dgat, Fasn, Scd1, and Acc in AAV-Vpr compared to AAV-GFP mice on a high-fat diet. (H) Increased mRNA expression of Ppara in AAV-Vpr compared to AAV-GFP mice on a high-fat diet. (I) Increased mRNA expression of Cpt1, Aox, Ehhadh, Acc2, and Lcad in AAV-Vpr compared to AAV-GFP mice on a high-fat diet. (J) Increased mRNA expression of MTP in AAV-Vpr compared to AAV-GFP mice on a high-fat diet. n = 5 in each group. Two-tailed, unpaired t-tests for unequal variance were used. P < .05 was considered significant. Values mean ± SE. *P < .05, **P < .01, ***P < .001: AAV-GFP vs AAV-Vpr.
Abbreviations: AAV, adeno-associated virus; GFP, green fluorescent protein; Vpr, viral protein R.
Discussion
We previously demonstrated that circulating and systemic expression of HIV-1 Vpr alters AT, liver and lipid and glucose metabolism in mice [14, 17]. Given the unique pathology observed in PWH and MASLD, we sought to define a tissue-specific role for Vpr in the development of hepatic steatosis, using a mouse model of liver-specific Vpr expression. Our results confirm and expand upon our previous findings [14], demonstrating that hepatic Vpr expression promotes glucose intolerance and has lipogenic effects that synergize with the metabolic stress of a HFD to induce IHTG accumulation. These data underscore the significant contribution of Vpr (and therefore HIV) to dysregulation of lipid metabolism, and potentially MASLD development, in PWH [14, 17-20], in whom additional factors, including unhealthy diet, adverse effects of ART, and other metabolic stressors likely synergize with HIV to promote or exacerbate hepatic steatosis [24, 25].
While it is well-established that HFD can independently induce hepatic steatosis in animal models, the development of MASLD in PWH is likely influenced by a combination of factors, including both viral and environmental or metabolic stressors. In this study, we aimed to disentangle the contributions of HIV-1 Vpr expression from those of diet-induced hepatic steatosis. To address the potential confounding effects of HFD alone, we included control experiments using nonvector (no AAV) mice fed a HFD. These control mice exhibited metabolic and hepatic phenotypes comparable to those of the AAV-GFP group. Although the data from nonvector controls are not shown in this manuscript, they support our conclusion that a synergistic effect on hepatic steatosis requires both HFD and hepatic expression of HIV-1 Vpr and suggest that the AAV GFP vector itself does not significantly contribute to hepatic steatosis. Hence, our data suggest that Vpr expression results in significantly greater IHTG and lipogenic gene expression compared to HFD-fed controls without Vpr. Thus, viral proteins, such as Vpr, and dietary or metabolic factors, such as HFD, likely combine to promote MASLD in PWH.
In addition to hepatic effects, we identified upregulation of inflammatory markers (Il6 and Il10) in the perigonadal adipose depot. We previously showed that mice expressing Vpr produced by a transgene under control of the PEPCK promoter (and hence expressed in liver, AT, and kidneys) is secreted into the circulation [17]—it is possible that AAV-Vpr expressed in the liver is similarly secreted and provokes inflammation in other tissues. These data contribute to our understanding of how Vpr may promote metabolic dysfunction beyond the liver.
Another key finding of this study is the glucose intolerance observed in AAV-Vpr mice on both regular chow and HFD. This systemic manifestation of liver-specific Vpr expression likely involves induction of hepatic insulin resistance, which is a multifactorial process. Disrupted hepatic lipid metabolism, local and systemic inflammatory signals, and alterations in AT function can all contribute to impaired insulin action. These data suggest that Vpr expression in the liver may serve aa a central node affecting multiple organs involved in glucose homeostasis [14, 17].
Energy balance did not differ between AAV-Vpr and control AAV-GFP mice, indicating that Vpr-induced glucose intolerance can occur independent of changes in whole-body energy expenditure or physical activity. Taken together with our previous studies in mice with more global delivery of Vpr, these data highlight the ability of Vpr to directly modulate an array of metabolic pathways (especially those related to hepatic lipid metabolism) through transcriptional coregulation [14, 17].
These data also demonstrate a critical mechanistic interaction between the direct effect of Vpr on the liver and that of HFD in inducing hepatic steatosis. On regular chow, there were no significant changes in the expression of genes involved in de novo lipogenesis, fatty acid oxidation, or lipid transport. However, the addition of high-fat feeding strikingly upregulated the expression of genes involved in de novo lipogenesis (including the master regulators Srebp1 and Chrebp, as well as those encoding the critical enzymes Fasn, Dgat, and Scd1), which would promote the synthesis of fatty acids and formation of IHTG. Conversely, genes involved in fatty acid oxidation (Pparα, Cpt1α, and genes related to β-oxidation and mitochondrial fatty acid metabolism) were significantly downregulated in AAV-Vpr mice on HFD. Finally, expression of Mttp was diminished in AAV-Vpr mice on HFD, which would impair export of VLDL-TG from the liver and promote lipid accumulation within hepatocytes, initiating or exacerbating hepatic steatosis. A notable consequence of this inhibitory effect on VLDL-TG export is the finding that, despite the significant IHTG accumulation, there was no elevation in plasma TG concentrations.
Collectively, these findings are consistent with our previous studies in the transgenic Vpr and pharmacologic synthetic Vpr mouse models [14], with 1 exception: we previously found that circulating plasma TG concentrations were increased in the transgenic and pharmacologic Vpr mouse models. However, this was in the context of more global delivery or expression of Vpr, and the elevated plasma TG despite inhibition of microsomal triglyceride transfer protein activity was due to the additional effect of Vpr to inhibit lipoprotein lipase activity in adipocytes [14]. Lipoprotein lipase activity is presumably normal in the current AAV-Vpr model, since the expression of Vpr is limited to the liver, accounting for the lack of increased circulating TG concentrations in these mice.
This study has significant implications for understanding the pathogenesis of MASLD in PWH. Namely, HIV-induced perturbations in lipid metabolism likely directly contribute to the high prevalence and severity of MASLD in PWH [3-10], interacting with traditional risk factors such as age, obesity, and diabetes to promote disease. Whether and how the effects of Vpr on lipid metabolism might be modulated by other environmental or metabolic factors, such as the gut microbiome, which is known to be altered in PWH and/or by ART [5, 25-27], is yet to be explored.
A limitation of our study is the use of male mice only, which may not be generalizable to female mice. Future studies should include female mice to assess potential sex differences in MASLD development and more comprehensive models incorporating ART or active viral replication to better simulate the conditions in PWH. Also, exploring molecular pathways by which Vpr and other HIV-1 products contribute to hepatic dysfunction will be critical for understanding its full impact on metabolic health in PWH and in developing potential therapeutic strategies to combat metabolic diseases associated with HIV.
Another limitation of our study is that body composition was assessed at different ages for the regular chow-fed and HFD-fed mice. This discrepancy arose because the metabolic effects of HFD require a longer duration of exposure to manifest, necessitating a later time point for evaluation. While this design allowed us to capture the progressive metabolic changes induced by HFD and the interaction with Vpr expression, it also introduces a variable of age and exposure duration that may influence direct comparisons between diet groups. Future studies with synchronized timelines or longitudinal assessments would help clarify these effects more precisely.
Additionally, we recognize that glucose tolerance tests primarily assess peripheral insulin sensitivity, mainly reflecting skeletal muscle glucose uptake, rather than hepatic insulin action. The hyperinsulinemic-euglycemic clamp technique, which was not performed in this study due to technical limitations, would offer a more precise and tissue-specific evaluation of insulin sensitivity and will be an important focus for future research. To partially address hepatic insulin resistance, we measured the expression of gluconeogenic genes Pck1 and G6pc, which were significantly elevated in HFD-fed Vpr mice, supporting a role for hepatic insulin resistance in this model (Fig. 4G).
In conclusion, our findings provide evidence that Vpr expression in the liver exacerbates dietary-induced metabolic dysfunction, particularly in the context of HFD. This highlights the potential for viral proteins to promote or exacerbate MASLD in PWH, with systemic inflammation and insulin resistance contributing to the overall pathogenesis. Future studies will need to investigate sex differences, measure serum Vpr in newer animal models, and include broader viral models to understand the full scope of viral protein contributions to metabolic dysfunction.
Acknowledgments
The authors thank the BCM Advanced Technology Cores for use of the following facilities: Gene Vector Core (where AAV-Vpr and AAV-GFP constructs were generated by Austin Seal Garrett and Kazuhiro Oka), Pathology and Histology Core, and Mouse Metabolism and Phenotyping Core.
Contributor Information
Neeti Agarwal, Division of Diabetes, Endocrinology, and Metabolism, Baylor College of Medicine, Houston, TX 77030, USA.
Pradip Saha, Division of Diabetes, Endocrinology, and Metabolism, Baylor College of Medicine, Houston, TX 77030, USA.
Claudia E Ramirez Bustamante, Division of Diabetes, Endocrinology, and Metabolism, Baylor College of Medicine, Houston, TX 77030, USA.
Sean M Hartig, Division of Diabetes, Endocrinology, and Metabolism, Baylor College of Medicine, Houston, TX 77030, USA; Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA.
Mark A Herman, Division of Diabetes, Endocrinology, and Metabolism, Baylor College of Medicine, Houston, TX 77030, USA.
Ashok Balasubramanyam, Division of Diabetes, Endocrinology, and Metabolism, Baylor College of Medicine, Houston, TX 77030, USA; Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030, USA.
Jordan E Lake, Email: Jordan.E.Lake@uth.tmc.edu, Division of Infectious Diseases, McGovern Medical School at UTHealth Houston, Houston, TX 77030, USA.
Funding
This work was funded by National Institutes of Health RO1 grant DK126047 (J.E.L.) and by the Rutherford Fund for Diabetes Research.
Author Contributions
A.B., S.M.H., J.E.L., and N.A. conceptualized the study. N.A., A.B., and S.M.H. designed experiments. N.A. and P.S. performed experiments. C.E.R.B. helped with the experiment. N.A., A.B., J.E.L., and S.M.H. wrote the manuscript with editorial input from all authors. All authors provided interpretations of the data. All authors have approved the submitted manuscript.
Disclosures
The authors declare no conflicts of interest.
Data Availability
Some or all datasets generated during and/or analyzed during the current study are not publicly available but are available from the corresponding author on reasonable request.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
Some or all datasets generated during and/or analyzed during the current study are not publicly available but are available from the corresponding author on reasonable request.