PPAR agonists as add-on treatment with metformin in management of type 2 diabetes: a systematic review and meta-analysis

The combination of metformin and the peroxisome proliferator-activated receptors (PPAR) agonists offers a promising avenue for managing type 2 diabetes (T2D) through their potential complementary mechanisms of action. The results from randomized controlled trials (RCT) assessing the efficacy of PPAR agonists plus metformin versus metformin alone in T2D are inconsistent, which prompted the conduct of the systematic review and meta-analysis. We searched MEDLINE and EMBASE from inception (1966) to March 2023 to identify all RCTs comparing any PPAR agonists plus metformin versus metformin alone in T2D. Categorical variables were summarized as relative risk along with 95% confidence interval (CI). Twenty RCTs enrolling a total of 6058 patients met the inclusion criteria. The certainty of evidence ranged from moderate to very low. Pooled results show that using PPAR agonist plus metformin, as compared to metformin alone, results in lower concentrations of fasting glucose [MD = − 22.07 mg/dl (95% CI − 27.17, − 16.97), HbA1c [MD = − 0.53% (95% CI − 0.67, − 0.38)], HOMA-IR [MD = − 1.26 (95% CI − 2.16, − 0.37)], and fasting insulin [MD = − 19.83 pmol/L (95% CI − 29.54, − 10.13)] without significant increase in any adverse events. Thus, synthesized evidence from RCTs demonstrates the beneficial effects of PPAR agonist add-on treatment versus metformin alone in T2D patients. In particular, novel dual PPARα/γ agonist (tesaglitazar) demonstrate efficacy in improving glycaemic and lipid concentrations, so further RCTs should be performed to elucidate the long-term outcomes and safety profile of these novel combined and personalized therapeutic strategies in the management of T2D. PROSPERO registration no. CRD42023412603.


Selection criteria
Any randomized control trial (RCT) enrolling adult patients with type 2 diabetes assessing the efficacy of any PPAR agonists plus metformin versus metformin alone was eligible for inclusion.RCTs in pediatric population or observational study designs were not eligible for inclusion.There were no restrictions on the inclusion according to the language of the publication, location, or date of study.

Search methods
A comprehensive and systematic search of PubMed and EMBASE databases was performed from inception until March 29, 2023.The complete search strategy for the two databases is illustrated in "Supplementary Appendix".There were no limits for language.Furthermore, references of relevant review articles and included studies were hand searched to identify additional eligible studies.

Data collection and analysis
All the citations obtained from the search was imported into EndNote software 29 .The duplicate citations were removed using the deduplication function in EndNote program.All the unique citations post deduplication were uploaded into Rayyan citation manager 30 .Two blinded review authors independently reviewed all titles, abstracts, and full-text reports to determine the eligibility of each reference for the inclusion in the systematic review as per the inclusion criteria using the Rayyan citation manager.Any disagreement in the inclusion was reviewed by the senior authors and resolved by consensus.

Data extraction and management
Two review authors independently extracted data using a paper based standardized data extraction form from all included studies.Data were collected on study characteristics (study design, setting), participant characteristics (number of participants enrolled, age,), intervention: characteristics (PPAR agonist, dose, route, administration schedule, and associated therapies) and outcomes.We did not consider any imputation methods for missing data.We had plans to contact the corresponding author in case of unextractable data.However, following standard approaches we were able to extract data from all reported outcomes.All abstracted data were entered into the Review Manager Package 31 .

Assessment of risk of bias in included studies
The risk of bias in the included studies was assessed using the Cochrane Risk of Bias assessment tool for RCT 32 .This tool includes the assessment of the method of randomization, allocation concealment, performance bias, detection bias, attrition bias, reporting bias, and any other bias.The overall certainty of evidence was assessed and summarized using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) method.This method separates the quality of evidence based on the risk of bias, inconsistency of results, indirectness of evidence, imprecision, and reporting bias 33 .

Assessment of heterogeneity and reporting biases
Heterogeneity between pooled studies was assessed using the I 2 statistic.An I 2 value of 0-40% might not be important; 30-60% may represent moderate heterogeneity, 50-90%: may represent substantial heterogeneity; and 75-100% considerable heterogeneity 28 .

Statistical analysis
All continuous data prior to analysis were converted into the same metric using the online Omni Health Calculator 34 .All analyses were performed following the intention-to-treat principle.In studies with multiple arms, we divided in half the subjects in the control groups when comparing against experimental arms.Continuous data were summarized as mean difference (MD) along with 95% confidence interval (CI) for each study.Dichotomous data were summarized as risk ratio (RR) along with a 95% CI for each study.When appropriate, summary estimates from individuals studies were pooled under a random-effects model using the DerSimonian-Laird approach outlined a priori in a protocol 35 .We decided a priori to use random effects model as the model of choice because it is more conservative compared with fixed effects and also incorporates the between-study variance into the calculation.We planned for stratified analysis by PPAR agonist type only.We did not plan for any other subgroup analysis, meta-regression or assessment of publication bias.6][37][38][39][40][41][42][43][44][45][47][48][49][50][51] .The mean FG was significantly lower in patients treated with metformin plus PPAR agonist compared to patients treated with metformin alone (MD = − 22.07 mg/dl, 95% CI = − 27.17, − 16.97; p < 0.001).Heterogeneity among pooled RCTs was substantial (I 2 = 83%).The overall certainty in the estimate was low (Table 2).
There was no significant difference between the subgroups (p = 0.82).
There was no significant difference between the subgroups (p = 0.10).
There was no significant difference between the subgroups (p = 0.44).
There was no significant difference between the subgroups (p = 0.10).
There was a significant difference between the subgroups (p = 0.009).
There was no significant difference between the subgroups (p = 0.97).
There was a significant difference between the subgroups (p = 0.008).
There was no significant difference between the subgroups (p = 0.32).
There was a significant difference between the subgroups (p = 0.0001).
There was a significant difference between the subgroups (p = 0.003).
There was no significant difference between the subgroups (p = 0.64).
There was no significant difference between the subgroups (p = 0.41).
Table 2. Summary of findings.*The risk in the intervention group (and its 95% confidence interval) is based on the assumed risk in the comparison group and the relative effect of the intervention (and its 95% CI).CI: confidence interval; MD: mean difference; RR: risk ratio.GRADE Working Group grades of evidence.High certainty: we are very confident that the true effect lies close to that of the estimate of the effect.Moderate certainty: we are moderately confident in the effect estimate: the true effect is likely to be close to the estimate of the effect, but there is a possibility that it is substantially different.Low certainty: our confidence in the effect estimate is limited: the true effect may be substantially different from the estimate of the effect.Very low certainty: we have very little confidence in the effect estimate: the true effect is likely to be substantially different from the estimate of effect.Explanations.a Of all the included studies, the reporting of the method of randomization sequence generation and allocation conceal is unclear for several studies.b Heterogeneity between studies is high.c Confidence intervals for the pooled estimate are wide.www.nature.com/scientificreports/Heterogeneity among pooled RCTs was not important (I 2 = 14%).The overall certainty in the estimate was moderate (Table 2).
There was no significant difference between the subgroups (p = 0.30).
There was no significant difference between the subgroups (p = 0.31).
All results from the subgroup analyses by the agent are reported in the supplementary material.

Discussion
The findings from our systematic review and meta-analysis, based on our knowledge, represent the largest body of synthesized evidence to date assessing the outcomes of metformin treatment alone versus combined treatment of metformin with PPAR agonists.The pooled results show that, on average, combination treatment with PPAR agonists compared with metformin alone is associated with significantly improved glycemic control in patients with type 2 diabetes.Specifically, the use of PPAR agonists plus metformin results in significantly lower concentrations of fasting glucose, hemoglobin A1c, fasting insulin, and HOMA-IR as compared to metformin treatment alone.In addition, the effect of combination treatment was consistent across all PPAR agonists types including PPARγ activators, pioglitazone and rosiglitazone, and dual PPARα/γ activator, tesaglitazar.These findings are in line with the previous studies that demonstrate the epidemiological and biological plausibility of these results.The beneficial effects of PPARα and PPARγ activation on glycemic control happens primarily by increasing insulin sensitivity and preserving beta-cell function 16,54 and the activation of PPARγ improves insulin sensitization and glucose uptake 7,18 .Treatment with PPARγ agonists, TZDs, effectively lowers HbA1c concentrations by about 1% as monotherapy and improves insulin sensitivity in patients with type 2 diabetes 55 .Furthermore, the pioglitazone treatment lowered concentrations of fasting glucose, insulin, and HbA1c in type 2 diabetic patients 56 , while another TZD, rosiglitazone, improved overall glucose tolerance and increased insulin sensitivity in patients with impaired glucose tolerance and type 2 diabetes 57 .Previous studies have also reported decreased HOMA-IR index, glucose, insulin, and HbA1c concentrations in diabetic patients upon an addition of rosiglitazone 23 and pioglitazone 25 to metformin treatment.Also, rosiglitazone provided more durable glycemic control than metformin or sulfonylurea 58 .Similarly, another study showed that the addition of pioglitazone to metformin-treated type 2 diabetic patients decreased HbA1c and HOMA-IR 42 as well as fasting insulin concentrations as compared with the sulfonylurea plus metformin group 59 .
The results of our meta-analysis demonstrated that treatment with PPARα/γ agonist, tesaglitazar, plus metformin reduced triglyceride (TG) concentrations in patients with type 2 diabetes, as compared to metformin   www.nature.com/scientificreports/treatment alone.However, treatment with TZDs (PPARγ agonists) plus metformin did not significantly affect TG concentrations.This is in line with the findings that the combined treatment of PPARα/γ agonist muraglitazar with metformin led to more enhanced effect in reducing TG concentrations as compared to the combined treatment of metformin with TZD agent pioglitazone 26 .Furthermore, results from recent clinical trials demonstrated that saroglitazar therapy decreased triglyceride concentrations by 45% as well as reduced concentrations of other atherogenic lipids, including TC, LDL-C, and VLDL-C 19,60 .It was also found that saroglitazar treatment improved lipid profile, including reduced TG, LDL-C, VLDL-C, TC, and increased HDL-C concentrations, in patients with type 2 diabetes receiving background metformin therapy 61 .The combined treatment of saroglitazar and metformin also resulted in a greater reduction of TG concentrations as compared to patients with type 2 diabetes who were treated with fenofibrate plus metformin 21 .
In addition to reduced triglyceride concentrations, our findings indicate that adding PPARα/γ activator (tesaglitazar) to metformin treatment does not significantly affect the concentrations of TC and LDL-C as compared to the patients with type 2 diabetes who were treated with metformin only.However, our meta-analysis demonstrates increased concentrations of total cholesterol and LDL-C upon treatment with PPARγ activators (TZDs) plus metformin vs metformin treatment alone.The results of the subgroup analysis per agent, showed that rosiglitazone plus metformin increased concentrations of TC and LDL-C, while pioglitazone plus metformin significantly affected TC concentrations only.This is in contrast to the previous studies which demonstrated that rosiglitazone has no significant effect on TG concentrations, while pioglitazone reduced TG and LDL particle size/concentrations 62 .Furthermore, it was reported that treatment with rosiglitazone plus metformin reduced concentrations of TG and TC 23 , while an addition of pioglitazone to metformin-treated patients with type 2 diabetes decreased TG, but increased HDL-C concentrations 59 .Since it was reported that metformin treatment itself reduces LDL-C concentrations in patients with type 2 diabetes 62,63 , it is possible that upon adding TZDs to metformin, the concentrations of LDL-C and/or TC concentrations increase as observed in our meta-analysis, which might be in line with the adverse effects of TZDs on the cardiovascular system.In line with our results, it was suggested that the potential difference in the risk of myocardial infarction between pioglitazone and rosiglitazone may lie in their different effects on lipoproteins concentrations, with pioglitazone demonstrating more favorable effects (TG decrease, HDL-C increase, with no effect on LDL-C or TC) than rosiglitazone (no effect on TG concentrations, HDL-C increase, but increases in LDL-C and TC concentrations) 64,65 .The pooled results as well as the results from the subgroup analysis according to the type of PPAR agonist showed the beneficial effects of the combined treatment of PPARα/γ or PPARγ agonists plus metformin vs metformin alone on HDL-C concentrations.This is in line with the previous studies, which also showed that increased HDL-C concentrations upon activation of PPARα 7,18 and PPARγ receptors 59,66 .
Furthermore, our findings also indicated the beneficial effects of combined treatment of PPARγ agonists with metformin, which decreased systolic and diastolic blood pressure in patients with type 2 diabetes.This is in line with previous reports indicating that the activation of PPARγ lowers systemic blood pressure 7,18,22,45 .
Another study also showed a reduction of systolic and diastolic blood pressure at 12 months of combined treatment with rosiglitazone and metformin, which correlated with HOMA-IR index, indicating that rosiglitazone can decrease blood pressure and that the enhancement of insulin sensitivity is associated with the reduction of blood pressure 23 .
Our findings showed that the concentrations of high-sensitivity C-reactive protein (hsCRP) were decreased following the combined treatment of PPARγ activators (TZDs) with metformin as compared to metformin treatment alone.This is in line with the previous studies, showing decreased concentrations of inflammation and cardiovascular risk markers, such as CRP, in obese and type 2 diabetic patients with TZD intervention 67 .Additionally, pioglitazone treatment significantly reduced CRP 56,68 and hsCRP concentrations 52,59 .The addition of rosiglitazone to metformin resulted in reduced concentrations of hsCRP as compared to type 2 diabetes patients who were treated only with metformin 22,45 .It was also reported that the combined treatment of PPARα/γ agonist muraglitazar with metformin led to a more enhanced effect in reducing hsCRP concentrations as compared to combined treatment of metformin with pioglitazone.However, our results demonstrated that there was no significant effect on hsCRP concentrations when another agent from this class of dual PPARα/γ agonist, tesaglitazar, was added to metformin treatment.
Our results of overall and subgroup analysis showed that there were no significant side effects associated with the addition of any PPAR agonist to metformin in patients with type 2 diabetes as compared to metformin, which is contrast to a few studies reporting an increased risk of adverse events associated with the use of PPAR agonists 19,58,65,69,70 .However, most studies reported either any adverse event or gastrointestinal toxicities, so we could not compare the cardiac and other toxicities possibly associated with addition of PPAR agonists.
Metformin treatment is associated with a high incidence of gastrointestinal (GI) side effects 62 .Strikingly, our meta-analysis showed that the risk of GI events was reduced after adding PPARγ activator (TZDs) to metformin treatment, while this beneficial effect was not observed upon combined treatment with dual PPARα/γ activator vs metformin treatment alone.
There are a several limitations to this systematic review and meta-analysis.These limitations primarily relate to the conduct and reporting of individual RCTs included here, which may possibly affect the overall results.For example, the overall methodological quality of evidence ranged from very low to moderate due to risk of bias and heterogeneity in pooled estimates.The risk of bias assessment may possibly be a function of reporting and not necessarily conduct.Similarly, the reasons for heterogeneity could be multifactorial, including difference in primary outcomes, study duration, type of PPAR agonist, metformin and PPAR dosing across pooled studies.Most included RCTs did report the sample size assessment details, which possibly reduces the chance of random error, and we suspect that, given the consistency of effects observed across all glycemic outcomes, the results are possibly not influenced by random error and risk of bias.Another important issue limitation relates to generalizability.All RCTs in this systematic review assessed the efficacy of either PPARγ or α/γ agonists and www.nature.com/scientificreports/therefore these findings are possibly limited to these specific types only and may not necessarily apply to PPARα or other PPAR agonists.However, our search did not find any RCTs assessing the efficacy of PPARα or other PPAR agonists.Once such RCTs are available this systematic review and meta-analysis will require an update.Nevertheless, despite these limitations, the impact of combination treatment with PPAR agonist compared with metformin alone in patients with type 2 diabetes is plausibly strong.
In conclusion, to the best of our knowledge, this meta-analysis is the largest body of synthesized evidence to date that assesses the outcomes of metformin treatment alone versus combined metformin and PPAR agonists in patients diagnosed with type 2 diabetes.Our findings indicate the beneficial effects of add-on treatment with PPAR agonist on glycemic control, reduced HbA1c concentrations, and ameliorated insulin resistance as compared to monotherapy with metformin.In addition, this combination also showed the favorable effects on type 2 diabetes-associated traits, including hypertension, increased concentrations of inflammatory markers, and dyslipidemia.Our meta-analysis demonstrated more favorable effects of combined metformin treatment with PPARα/γ activator (tesaglitazar) on lipid profile by lowering the concentrations of TG, increasing HDL-C concentrations and not increasing the concentrations of LDL-C and TC observed upon adding TZDs to metformin treatment.This might be in line with the adverse effects of TZDs on the cardiovascular system.However, our results also indicate that adding TZDs to metformin treatment results in a more favorable safety profile by reducing the number of GI adverse events as compared to type 2 diabetic patients who were treated with metformin only.Thus, it is crucial to consider the beneficial as well as the potential adverse events, such as gastrointestinal and cardiovascular events, which are related to use of the combination of metformin and PPAR agonists that would require monitoring and the potential adjustment of the prescribed medication or its dose.Further studies are warranted to elucidate the long-term outcomes and optimal usage of combined metformin and PPAR agonist in the management of type 2 diabetes, with continued research exploring optimal dosing regimens, long-term effects, and personalized treatment approaches.In addition, the potential for novel PPAR agonists with improved safety profiles warrants further investigation.