Plasma Prostaglandin E2 Metabolite Levels Predict Type 2 Diabetes Status and One-Year Therapeutic Response Independent of Clinical Markers of Inflammation

Over half of patients with type 2 diabetes (T2D) are unable to achieve blood glucose targets despite therapeutic compliance, significantly increasing their risk of long-term complications. Discovering ways to identify and properly treat these individuals is a critical problem in the field. The arachidonic acid metabolite, prostaglandin E2 (PGE2), has shown great promise as a biomarker of β-cell dysfunction in T2D. PGE2 synthesis, secretion, and downstream signaling are all upregulated in pancreatic islets isolated from T2D mice and human organ donors. In these islets, preventing β-cell PGE2 signaling via a prostaglandin EP3 receptor antagonist significantly improves their glucose-stimulated and hormone-potentiated insulin secretion response. In this clinical cohort study, 167 participants, 35 non-diabetic, and 132 with T2D, were recruited from the University of Wisconsin Hospital and Clinics. At enrollment, a standard set of demographic, biometric, and clinical measurements were performed to quantify obesity status and glucose control. C reactive protein was measured to exclude acute inflammation/illness, and white cell count (WBC), erythrocyte sedimentation rate (ESR), and fasting triglycerides were used as markers of systemic inflammation. Finally, a plasma sample for research was used to determine circulating PGE2 metabolite (PGEM) levels. At baseline, PGEM levels were not correlated with WBC and triglycerides, only weakly correlated with ESR, and were the strongest predictor of T2D disease status. One year after enrollment, blood glucose management was assessed by chart review, with a clinically-relevant change in hemoglobin A1c (HbA1c) defined as ≥0.5%. PGEM levels were strongly predictive of therapeutic response, independent of age, obesity, glucose control, and systemic inflammation at enrollment. Our results provide strong support for future research in this area.


Plasma PGEM Is a Strong Predictor of T2D Disease Status
Plasma PGEM levels were, on average, two-fold higher in the T2D group as compared to ND controls (51.6 ± 30.4, ND vs. 101.5 ± 39.1, T2D; p < 0.0001) (Table 1 and Figure 3A). There were no statistically significant correlations between plasma PGEM and BMI, HbA1c, WBC, and triglycerides, and only a weak correlation with ESR (Table 1). The rate-limiting step in PGE 2 production is catalyzed by cyclooxygenase (COX) enzymes, and aspirin is a COX-1 inhibitor [39]. We found no difference in mean PGEM levels between those in the T2D group who reported low-dose daily aspirin use (n = 54) and those who did not (n = 71) ( Figure 3B). Eicosapentaenoic acid (EPA) is an omega-3 polyunsaturated fatty acid that competes with arachidonic acid for the same site in plasma membrane phospholipids [10]. We found no difference in mean PGEM between those in the T2D group who reported omega-3/fish oil supplement use (n = 37) and those who did not (n = 95) ( Figure 3C). A logistic regression analysis including age, sex, BMI, WBC, triglycerides, aspirin use, and PGEM revealed PGEM as the strongest predictor of T2D diagnosis (p < 0.0001) ( Figure 3D and Table 2) (ESR was not included in this analysis as it was strongly associated with WBC (p < 0.0001; R 2 = 0.12).
( Figure 3C). A logistic regression analysis including age, sex, BMI, WBC, triglycerides, aspirin use, and PGEM revealed PGEM as the strongest predictor of T2D diagnosis (p < 0.0001) ( Figure 3D and Table 2) (ESR was not included in this analysis as it was strongly associated with WBC (p < 0.0001; R 2 = 0.12).

T2D Patients with High Plasma PGEM Levels Have Significantly Worse Blood Glucose Control One-Year Post-Enrollment
T2D subjects were assessed by chart review one year following the study enrollment, and their percent change in HbA1c was calculated. Seven subjects were lost to follow-up, and three additional subjects whose HbA1c increased more than 4% were excluded due to non-compliance. The final analysis included 45 subjects with a clinically significant reduction in HbA1c (≥0.5%) one-year post-enrollment and 77 without (n = 122).
As there is no clinical threshold for plasma PGEM, the median PGEM level from all 132 T2D patients (92.96 pg/mL) was used to classify T2D subjects in either a "low" or "high" PGEM group for follow-up analyses ( Figure 4A). On average, T2D subjects with low plasma PGEM exhibited a 0.6% decrease in HbA1c: a statistically significant difference from those in the high PGEM group, where no change in mean HbA1c was observed (p = 0.0019) ( Figure 4B). In total, 47.5% of T2D patients with low plasma PGEM levels achieved a clinically significant reduction in HbA1c (≥0.5%) over 1 year ( Figure 4C). Conversely, only 25.8% of T2D patients with high plasma PGEM were able to achieve a clinically significant reduction in HbA1c over 1 year ( Figure 4C).

T2D Patients with High Plasma PGEM Levels Have Significantly Worse Blood Glucose Control One-Year Post-Enrollment
T2D subjects were assessed by chart review one year following the study enrollment, and their percent change in HbA1c was calculated. Seven subjects were lost to follow-up, and three additional subjects whose HbA1c increased more than 4% were excluded due to non-compliance. The final analysis included 45 subjects with a clinically significant reduction in HbA1c (≥0.5%) one-year post-enrollment and 77 without (n = 122).
As there is no clinical threshold for plasma PGEM, the median PGEM level from all 132 T2D patients (92.96 pg/mL) was used to classify T2D subjects in either a "low" or "high" PGEM group for follow-up analyses ( Figure 4A). On average, T2D subjects with low plasma PGEM exhibited a 0.6% decrease in HbA1c: a statistically significant difference from those in the high PGEM group, where no change in mean HbA1c was observed (p = 0.0019) ( Figure 4B). In total, 47.5% of T2D patients with low plasma PGEM levels achieved a clinically significant reduction in HbA1c (≥0.5%) over 1 year ( Figure 4C). Conversely, only 25.8% of T2D patients with high plasma PGEM were able to achieve a clinically significant reduction in HbA1c over 1 year ( Figure 4C).

Discussion
In this study, we demonstrate that plasma PGEM shows promise as a circulating biomarker to assess the risk of T2D diagnosis and the efficacy of blood glucose management in individuals with T2D. Plasma levels of a stable metabolite of PGE2 were significantly higher in individuals living with T2D when compared to a control group. This finding is consistent with recent work from our group and others using small numbers of biosamples from obese, ND, and T2D subjects [18,[20][21][22]. These results with a larger clinical cohort both validate the previous findings, as well as reveal PGEM as a strong predictor of T2D disease status: even more so than validated clinical tests of systemic inflammation. Finally, for the first time, we discovered plasma PGEM was a strong predictor of T2D therapeutic response over the following year. Our findings provide strong evidence for further investigations into the role of PGE2 metabolites in diabetes pathogenesis and treatment response.

Discussion
In this study, we demonstrate that plasma PGEM shows promise as a circulating biomarker to assess the risk of T2D diagnosis and the efficacy of blood glucose management in individuals with T2D. Plasma levels of a stable metabolite of PGE 2 were significantly higher in individuals living with T2D when compared to a control group. This finding is consistent with recent work from our group and others using small numbers of biosamples from obese, ND, and T2D subjects [18,[20][21][22]. These results with a larger clinical cohort both validate the previous findings, as well as reveal PGEM as a strong predictor of T2D disease status: even more so than validated clinical tests of systemic inflammation. Finally, for the first time, we discovered plasma PGEM was a strong predictor of T2D therapeutic response over the following year. Our findings provide strong evidence for further investigations into the role of PGE 2 metabolites in diabetes pathogenesis and treatment response.
While obesity is a driver of T2D pathology, and changes in plasma PGEM could indicate glucolipotoxic metabolic and inflammatory dysfunction, we demonstrated no biologically relevant correlations to other biomarkers of obesity, inflammation, or insulin resistance, including BMI, WBC, ESR, and triglyceride levels ( Table 1, Figures 1 and 2). Logistic regression analysis of the data set, including age, sex, BMI, WBC, triglycerides, and aspirin use indicated the most significant predictor of T2D status was plasma PGEM, and subjects having plasma PGEM levels greater than 101.5 pg/mL had a 99% probability of a T2D diagnosis ( Figure 3D and Table 2).
Currently, recommendations by the American Diabetes Association (ADA) suggest that testing to assess risk for future diabetes in asymptomatic people should be considered in adults of any age who are overweight or obese and have one or more additional risk factors, including physical inactivity, first-degree relative with diabetes, women with gestational diabetes mellitus, hypertension, women with polycystic ovary syndrome, history of cardiovascular disease, and others [58]. However, current diagnostic tests are imperfect and prone to misclassification errors. HbA1c has not been validated for all populations [59] and can be confounded by structural variants in the hemoglobin molecule or alterations in red blood cell turnover. The use of plasma PGEM as an additional measure of T2D status could act as a secondary means of quantifying T2D risk assessment. The inclusion of plasma PGEM in this list of risk factors may capture high-risk individuals who may be otherwise go undiagnosed.
Historically, clinical values including HbA1c, intact proinsulin, adiponectin, and high sensitivity C-reactive protein have been suggested as biomarkers of β-cell failure and insulin resistance, although their overall useability is limited, as both specificity and context need to be considered [60]. Individualized treatments based on the precision understanding of an individual's disease process have garnered much enthusiasm. Ahlqvist et al., used cluster analysis to define five subgroups of individuals based on their diabetes characteristics and risk for developing diabetic kidney disease using six parameters (BMI, HbA1c, glutamic acid decarboxylase antibodies, and homeostatic model assessment of insulin resistance (HOMA-IR) and insulin secretion (HOMA-B) [61]. The results of this study suggested the need to identify additional biomarkers to improve sensitivity and precision in stratifying individuals with pre-diabetes and T2D. Recently, dihydroceramides have been shown to act as a potential biomarker for T2D [7]. In addition to predicting T2D disease status, PGEM is one of a few putative biomarkers that may also provide functional insight into β-cell health relevant to the present therapeutic landscape. Specifically, the expression of PGE 2 synthetic and signaling enzymes is higher in pancreatic islets isolated from T2D mice and human organ donors than in non-diabetic controls [9,10,14,21,41,51,[62][63][64], resulting in significantly elevated PGE 2 release [9,10]. In the β-cell, PGE 2 binds to the G z -coupled prostaglandin E 2 EP3 receptor (EP3) [13,40,[65][66][67], which, when activated, limits insulin secretion in response to glucose and glucagon-like peptide 1 receptor (GLP1R) agonists: a mechanism that actively contributes to the β-cell dysfunction of the disease [9,67]. GLP1-RAs are currently in wide use as first-or second-line T2D therapeutics, yet, despite the popularity of these drugs in the clinic, they do not have the same efficacy in all patients. With the known inhibitory effect of PGE 2 receptor antagonists on the efficacy of GLP1-RAs in preclinical models, this finding may be of great importance in clinical decision-making. One limitation is that we did not directly measure β-cell function by methods such as quantifying stimulated C-peptide levels; therefore, this possibility remains only theoretical.
Another limitation of the current observational study is it was neither adequately powered nor designed to assess the specific impact of PGEM levels on GLP1-RA efficacy. Future work must include trials of drug-naïve patients with T2D randomized to different classes based on plasma PGEM levels to determine if these could be used to help providers choose the drug that will work best for each patient.
We acknowledge several other limitations of this study. The population in the UWHC catchment area is primarily white/non-Hispanic; therefore, our results may not be representative of more diverse populations. This is an important limitation, as recent studies have found the appropriateness of common biomarkers of T2D risks differ, based on an individual's racial and ethnic background [68]. Second, as this was an observational study with the primary outcome being plasma PGEM, we did not control for time since T2D diagnosis or current T2D therapeutics. The ongoing management of the subjects' T2D during the 1 year follow-up period was not influenced in any way by this research study and, therefore, was based on "real-world" standard clinical care. Third, diet quantity/composition and physical activity can impact diabetes control, and in the current study, we did not have participants keep diet or exercise logs to quantify this potential confounder. Finally, as plasma PGEM in a T2D clinical cohort has not previously been studied, it will be necessary to identify and validate an appropriate clinical threshold if it is to be used as a biomarker for T2D therapeutic response. These additional considerations are important to note but fall outside the scope of this study.

Conclusions
In this clinical cohort study, we find plasma PGEM levels are an excellent predictor of T2D status and one-year therapeutic response, independent of known markers of inflammation, obesity, and T2D disease control. These findings were surprising, as hyperglycemia, dyslipidemia, and pro-inflammatory cytokines have all been shown to upregulate enzymes in the PGE 2 production pathway. Our results provide strong support for future research into plasma PGEM as an independent biomarker for T2D status and long-term disease control. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: Not applicable.