Effect of dolutegravir-based antiretroviral therapy on glycemic control in female mice

Categories: Disease & Virus

June 5, 2025

DTG-based ART in female mice is associated with a transient dysregulation of glycemic control, but not with excess weight gain or food intake

To assess metabolic alterations due to DTG-based ART, single-housed female C57Bl/6J mice were treated with either control, 1xDTG, or 5xDTG regimens once daily starting at 8 weeks of age. Their weekly food intake, body weight, and morning fasted blood glucose were recorded over 8 weeks. Female mice were selected for this study as female sex was one of the risk factors identified for DTG-related weight gain and we were interested in looking at glucose dysregulation as a risk factor for NTD.

Weekly food intake did not differ between treatments (Fig. 1A). Mice in all treatment arms gained weight at a similar rate. No differences in weight gain were observed between DTG-treated and control animals (Fig. 1B). Morning fasted (4-6h) blood glucose in the control animals remained at around 9mmol/L, while mildly elevated blood glucose was seen in weeks 2–4 of treatment in the 1xDTG treated mice and in weeks 1–3 in the 5xDTG treated mice in comparison to the controls (Fig. 1C), although none reached significance.

Overnight (13-15h) fasted blood glucose (measured prior to administration of glucose in the OGTT) rose over the course of the study more prominently in the 1xDTG-treated group, but also mildly in the 5xDTG group (Fig. 1D). In the 1xDTG group, overnight fasted glucose increased steadily between the period of 2 to 6 weeks, reaching a peak at 6 weeks and then declining to similar glucose levels as those seen in the control group by 8 weeks. Overnight fasted blood glucose was significantly higher in the 1xDTG group with a trend to higher in the 5xDTG group compared to the control group at 6 weeks. Notably, several animals in both the 1xDTG (46%, 6 of 13) and 5xDTG (33%, 5 of 15) groups developed hyperglycemia with blood glucose reaching above 10.4 mmol/L at least one time during the treatment window, with the peak number of hyperglycemic mice occurring after 6 weeks of treatment (Fig. 1E). No hyperglycemic mice were observed in the control group. We also measured overnight fasted glucose in mice at time of sacrifice, following 9 weeks of treatment. Glucose levels did not differ significantly between groups, although median glucose levels in the 1xDTG group were higher than controls (Fig. 1F).

Overnight fasted mice were also administered an oral glucose tolerance test (OGTT) at 2, 4, 6, and 8 weeks of treatment. There were no significant differences in the OGTT (Fig. 2A) or AUC to determine glucose clearance (Fig. 2B), between treatment arms at any time point in the study.

DTG treatment is associated with a progressive reduction in plasma leptin and an increase in corticosterone levels

As we observed a transient increase in overnight fasted glucose in the 1xDTG group and in some mice in the 5xDTG group, we next investigated changes to plasma hormones associated with regulation of glucose. We first measured insulin, the main glucoregulatory hormone, in overnight fasted plasma over 9 weeks of treatment. Insulin levels were similar between treatment groups, with a small non-significant increase seen at week 6 in the 5xDTG group and week 9 in the 1xDTG group (Sup. Fig. S1A, B).

Leptin is an adipocyte produced hormone with glucoregulatory function. Lower leptin secretion has been reported in DTG-treated adipocytes^29,38. Mean plasma leptin levels in overnight fasted mice were marginally lower in the 1xDTG group compared to control at week 2 and became significantly lower from week 4 onward reaching the lowest levels at week 6 and 8 (Fig. 3A). Leptin levels declined only mildly in the 5xDTG group, reaching significance only at the 6-week time point. Leptin levels remained lower in both DTG groups compared to controls at time of sacrifice at week 9 (Fig. 3B). To corroborate our observations of lower plasma leptin levels with DTG treatment, we quantified leptin expression in white adipose tissue collected at time of sacrifice following 9 weeks of treatment. Leptin mRNA levels in white adipose tissue were lower in both DTG-treated groups compared to controls, although this did not reach significance (Fig. 3C). There was a strong positive correlation between peripheral leptin levels and adipocyte leptin expression (r = 0.77, p < 0.0001, Fig. 3D).

Due to the glucoregulatory role of leptin through cortisol (corticosterone in mice),³⁹ we measured corticosterone levels in plasma following the overnight fast and calculated the difference in mean concentrations compared to controls. Corticosterone levels were mildly elevated during weeks 2 and 4 in 1xDTG mice, and progressively increased, reaching significance, over weeks 6 and 8 (Fig. 3E). Corticosterone levels in the 5xDTG group did not differ from the control group. A similar trend was observed at time of sacrifice at week 9, with corticosterone levels remaining significantly higher in the 1xDTG group compared to controls (Fig. 3F). We observed a strong negative correlation between corticosterone levels at week 9 and white adipose tissue leptin mRNA (r = -0.67, p < 0.0001, Fig. 3G), as well as plasma leptin levels (r = -0.45, p = 0.0035).

DTG treatment increases leptin receptor expression in muscle and liver.

Since we observed persistently low leptin levels with DTG treatment, even-though euglycemia was restored by week 8 and 9, we next examined if DTG-treated mice could have adapted to the lower leptin levels by upregulating leptin receptor expression. We observed significantly higher leptin receptor expression in the muscle for both DTG groups compared to controls (Fig. 4A), and a trend towards higher leptin receptor expression in the liver of the 1xDTG group (Fig. 4C). We observed a negative correlation between both muscle (Fig. 4B) and liver (Fig. 4D) leptin receptor and plasma leptin levels (r = -− 0.35, p = 0.026 for muscle; r = -− 0.59, p < 0.0001 for liver), with lower plasma leptin levels being associated with higher leptin receptor expression. Higher leptin receptor expression could increase leptin sensitivity and act as a compensatory mechanism in response to the lower leptin levels⁴⁰.

DTG-based ART exposure has a mild effect on hepatic and muscle gene expression.

Since we observed a transient hyperglycemia in the DTG-treated mice, we speculated that compensatory mechanisms may have contributed to restoring or maintaining euglycemia. The liver is the key regulator of endogenous glucose production in a fasted state and integrates peripheral and central endocrine and neural signals for maintenance of glucose homeostasis. We measured gene expression of proteins involved in gluconeogenesis, fatty acid catabolism, and lipogenesis in livers collected at sacrifice following 9 weeks of treatment. We hypothesized that a reduction in endogenous glucose production and/or fatty acid catabolism could potentially offset the mechanisms contributing to hyperglycemia and either restore euglycemia or protect mice from developing hyperglycemia. Means with 95% CI of mRNA levels for all genes assessed are shown in Table 1 for liver, Table 2 for muscle, and Supplemental Table S2 for brown adipose tissue, Table S3 for hypothalamus, and Table S4 for white adipose tissue.

Table 1 Hepatic gene expression after 9 weeks of treatment.

Table 2 Muscle gene expression after 9 weeks of treatment.

In the liver, we did not observe changes to the gluconeogenic phosphoenolpyruvate carboxykinase (Pepck) or glucose-6-phosphatase (G6p) between treatment arms, however both G6p and Pepck showed greater spread in the data in the DTG groups (Table 1). In pro-lipogenic genes, we saw a downregulation of hepatic fatty acid synthase (Fas) mRNA expression in both DTG groups that reached significance in the 5xDTG group (Fig. 5A). Diacylglycerol O-acyltransferase 1 (Dgat1) expression was also significantly downregulated in the 5xDTG group (Fig. 5B). No differences were observed in Acc1/2 (Fig. 5C, Table 1), Dgat2, or sterol regulatory element-binding protein 1C (Srebp1c) levels between groups (Table 1). No significant differences were observed in peroxisome-proliferator-activated receptor gamma coactivator 1-alpha (Pgc1a), peroxisome-proliferator-activated receptor alpha (Pparα), or Ppparγ (Table 1). We also observed a trend towards lower mRNA levels of insulin receptor substrate (Irs2) with DTG treatment, that reached significance in the 5xDTG group (Fig. 5D). Tdo, which controls the rate limiting step of the catabolism of tryptophan to kynurenines, was higher in the 1xDTG group although this did not reach significance (Table 1).

To examine if changes in liver mRNA levels translated to changes in protein levels, we quantified FASN, ACC1, and DGAT1 protein levels in liver lysates using Western blot. ACC1 and FASN were significantly lower in the 5xDTG group only (Fig. S2A and B). DGAT1 levels did not differ between treatment groups (Fig. S2C).

In muscle, we observed higher levels of Cd36 that reached significance in the 1xDTG group, higher levels of Irs1 in both DTG groups, and significantly lower levels of Pparγ in the 1xDTG group only (Fig. 5E-G). Levels of Glut4, Pparα and Acc1 were similar between groups (Table 2).

Other potential pathways that could stabilize glycemia include increased brown adipose lipid uptake and thermogenesis, and changes to hypothalamic energy balance. We did not observe changes to transcripts of uncoupling protein-1 (Ucp1), responsible for thermogenesis, nor of brown adipose tissue markers cell death-inducing DFFA-like effector A (Cidea) and type II iodothyronine deiodinase (Dio2) in brown adipose tissue (Table S2). In hypothalamic tissue, we observed a non-significant upregulation of orexigenic agouti-related peptide (Agrp) mRNA expression in both DTG-treated groups compared to control, suggesting a negative energy balance in some animals. No differences were observed in the levels of Pomc, Bdnf, or Trh (Table S3). As noted above, leptin (Lep) transcripts were downregulated in gonadal white adipose tissue in DTG-treated mice, although this did not quite reach significance (Fig. 3C). No differences were observed between groups in adiponectin, Pparγ, Fas, or Srebp1c levels in white adipose tissue (Table S4).

Differential regulation of metabolic genes in the liver of 5xDTG mice that maintained euglycemia.

The greater spread in the expression data observed in the DTG groups, prompted us to examine if gene expression patterns may differ by glycemia status (euglycemic vs. hyperglycemic). Expression data for all the genes analyzed in the liver were log transformed and the means displayed in a heat map stratified by treatment arm and glycemia state (Fig. 6A). Volcano plots were generated to visualize significant differences in gene expression between each treatment/glycemia state arm and controls, using a false discovery rate of 10% (Fig. 6B). Compared to controls, the greatest difference in expression patterns was observed in the euglycemic–5xDTG group. Euglycemic mice in the 5xDTG group had significant downregulation in the gluconeogenic genes Gck, Pepck, and G6p, as well as downregulation in Dgat1, Pparα, Acc1, Irs2, Insig2, and Fas (Fig. 6B top right panel). Pparα and Fas were also significantly downregulated in the euglycemic–1xDTG group (Fig. 6B top left panel). Tdo was the only gene significantly upregulated, with the upregulation seen in the hyperglycemic–5xDTG.

A similar analysis in muscle (Fig. 7) showed an upregulation of Cd36 and Lepr in both the euglycemic–5xDTG and the hyperglycemic–1xDTG groups. However, the hyperglycemic–1xDTG group also showed a downregulation of Pparγ and Glut4, suggesting perhaps that Cd36 and LepR upregulation may be protective, but only in the absence of Pparγ and/or Glut4 downregulation.

No hepatic lipid accumulation in DTG-treated mice

As we observed increased corticosterone and lower leptin combined with mild downregulation in gene expression of factors associated with lipogenesis (Fas, Acc1) with DTG treatment, we stained liver sections with oil-red-O (ORO) to assess triacylglycerol accumulation. We did not observe any differences in average lipid droplet size or ORO stain density (Fig. S3).

Source link