Combined Intervention With Pioglitazone and n-3 Fatty Acids in Metformin-treated Type 2 Diabetic Patients

Improvement of Lipid Metabolism

Jiri Veleba; Jan Kopecky Jr.; Petra Janovska; Ondrej Kuda; Olga Horakova; Hana Malinska; Ludmila Kazdova; Olena Oliyarnyk; Vojtech Skop; Jaroslava Trnovska; Milan Hajek; Antonin Skoch; Pavel Flachs; Kristina Bardova; Martin Rossmeisl; Josune Olza; Gabriela Salim de Castro; Philip C. Calder; Alzbeta Gardlo; Eva Fiserova; Jørgen Jensen; Morten Bryhn; Jan Kopecky Sr.; Terezie Pelikanova

Disclosures

Nutr Metab. 2015;12(52) 

In This Article

Discussion

We show here that a 6-month-combined intervention using a relatively low dose of pioglitazone and a dose of EPA + DHA, which is within the range that is approved for treatment of hypertriacylglycerolemia,[13] exerts additive beneficial effects on metabolism of both NEFA and triacylglycerols in T2D patients (Fig. 4). All the patients were already receiving metformin therapy and well compensated regarding glycemic control at the baseline, and most of them were obese, thus representing a typical population of patients treated for T2D.

In spite of the triacylglycerol-lowering effect of EPA + DHA, shown in many studies, including those in patients with T2D, as well as the hypolipidemic effects of thiazolidinediones (see Background), fasting serum triacylglycerol levels were not significantly affected by any of the studied interventions. It is likely that the background metformin therapy, which was shown to improve dyslipidemia in patients with T2D,[30] could mask the effect of the tested interventions. This is also consistent with the notion that most of the studies demonstrating the triacylglycerol-lowering effect of EPA + DHA in the patients with T2D were performed before the beginning of the metformin era as well as the use of thiazolidinedione therapy of these patients. Moreover, metformin could also mask the additive triacylglycerol-lowering effects of EPA + DHA in T2D dyslipidemic patients under statin therapy, which was observed before[31] but not in our study (not shown). Nevertheless, the additive improvement in metabolism of both NEFA and triacylglycerols by the combined intervention found using the meal test in this study (Fig. 4) suggests that increased intake of EPA + DHA could reduce the cardiovascular risk even in T2D patients treated with metformin. This complex effect of the combined intervention is of clinical relevance because increased postprandial triacylglycerolemia represents an independent risk factor of cardiovascular disease in T2D patients.[10,17] The mechanisms behind the effect of the combined intervention on metabolism of NEFA and triacylglycerols require clarification. It is likely that PPARα-mediated catabolism of fatty acids[12] and/or their trapping in adipose tissue, i.e., the biochemical activities that are possibly altered in the patients (reviewed in[32]), could contribute to the NEFA-lowering effect. Regarding the beneficial effect on metabolism of triacyglycerols, depression of the rate of VLDL-triacyglycerol secretion from the liver should be considered.[7,33]

That all the patients were well-controlled under metformin therapy could also affect other results of the study. First, it could explain why no effect on proteins linked to inflammation (anti – inflammatory cytokines, namely IL-1RA, IL-10, and pro-inflammatory cytokines, namely MCP-1, CRP, TNF-α, IL-6) was observed (Additional file 3 http://static-content.springer.com/esm/art%3A10.1186%2Fs12986-015-0047-9/MediaObjects/12986_2015_47_MOESM3_ESM.docx), since metformin is known to exert anti-inflammatory action.[3] Indeed cytokine and adhesion molecule concentrations (cVCAM-1, sICAM-1, sE-selectin, sPECAM-1) were similar to those reported for healthy male subjects of different ages.[34] Importantly, no deleterious effects of any of the interventions on the markers of oxidative stress (SOD, TBARS, ratio GSSG/GSH) were observed (Table 1), possibly, due to the antioxidant effect of metformin.[4] Similarly, also insulin secretion remained unaffected by the interventions (see Results, Postprandial metabolism).

Second, only limited additional benefits of pioglitazone and/or Omega-3 may be expected in metformin treated T2D patients. In fact, Omega-3 alone marginally impaired markers of glycemic control (HbA1c levels and fasting glycemia; Table 2) and glucose metabolism in the meal test (Fig. 4), but did not diminish insulin sensitivity (M) evaluated using a hyperinsulinemic-euglycemic clamp (Table 2). These results are compatible with a model in which EPA + DHA per se do not deteriorate glucose utilization when glucose represents the main energy fuel (Fig. 5, Clamp). However, when the supply of both carbohydrates and lipids is increased (e.g., during the meal test; Fig. 5, Fed), or when fatty acids liberated from adipose tissue represent the main energy fuel (Fig. 5, Fasting), glucose utilization is inhibited by multiple mechanisms involved in the Randle cycle (reviewed in[35]) reflecting the PPARα-mediated stimulation of fatty acid oxidation by EPA + DHA.[12] This would lead to the observed subtle deterioration of glucose metabolism by Omega-3. Metabolic changes in skeletal muscle, the main site of glucose utilization, probably play a major role. That fasted glycemia is selectively increased by Omega-3 (Table 2) could also reflect increased hepatic gluconeogenesis stimulated in face of enhanced fatty acid oxidation.[36] Both decreased postprandial metabolism of glucose (Fig. 4) and elevated glycemia in fasted state could contribute to raised HbA1c levels in the Omega-3 subgroup (Table 2). Thus, our results also help to clarify some controversies regarding the effects of EPA + DHA on glucose homeostasis in T2D patients (see Background).

Figure 5.

The effects of omega-3 fatty acids on glucose metabolism in insulin-sensitive tissues depend on energy fuels. When glucose serves as the major energy substrate (Clamp), glucose utilization is only marginally affected. With increased postprandial intake of both carbohydrates and lipids (Fed/postprandial), or when fatty acids (FA) serve as the major fuel (Fasting), β-oxidation is stimulated by EPA + DHA via PPARα-signaling,[12] which results in reduced glucose utilization (red dashed lines) by several mechanisms involving the Randle cycle (55). Inhibition of glucose oxidation at the level of pyruvate dehydrogenase (PDH) by acetyl-CoA (a) leads also to rerouting of pyruvate to anaplerosis (muscle) and/or gluconeogenesis (liver); citrate accumulation in the cytosol results in inhibition of glucose uptake (b) and inhibition of glycolysis (c) at the level of hexokinase (HK)

The negative effects of Omega-3 on glycemic control and glucose metabolism were prevented by Pio. Insulin sensitivity was increased by Pio&Omega-3, and tended to be improved by Pio compared with Omega-3 (Table 2), which is also in agreement with the induction of adiponectin by both Pio& Omega-3 and Pio (Table 1). These results were consistent with changes in metabolic flexibility to glucose evaluated using indirect calorimetry during the hyperinsulinemic-euglycemic clamp at week 24 (Fig. 3), since this parameter closely reflects whole-body glucose uptake.[27] Thus, all the interventions, and especially Pio& Omega-3, increased metabolic flexibility. These results are consistent with the above model and also with our previous study showing additive improvement in metabolic flexibility[20] and insulin sensitivity[7] by combined interventions using EPA + DHA and thiazolidinediones in dietary obese mice. In both the animal experiments and the present clinical trial, the robust PRCF analysis of RQ was used. This approach, which revealed here subtle differences in metabolic flexibility, has not been applied in humans before.

Few studies were conducted to characterize possible modulation of metabolic flexibility by EPA + DHA in T2D patients, and very little is known about the effects of combined interventions using EPA + DHA and pharmaceuticals. It has been shown that EPA + DHA administered as a 4-h lipid infusion resulted in a marginal improvement of metabolic flexibility without affecting insulin sensitivity.[37] Over a 9-week-period, dietary EPA + DHA exerted a transient improvement of glucose utilization followed by a shift from glucose to lipid catabolism, but the effect on metabolic flexibility is difficult to assess from these data since a relatively large volume (20 ml) of crude fish oil containing different lipid fractions besides EPA + DHA was used.[38] Thus, our study is unique regarding the use of a complex methodological approach including the indirect calorimetry, clamp as well as a meal test, which allowed us to demonstrate the additive improvements in metabolic flexibility to glucose, and namely in the postprandial lipid metabolism, by pioglitazone in combination with highly purified EPA + DHA.

Evaluation of serum levels of both pioglitazone and EPA + DHA increased the power of the study by controlling the adherence to the therapy, and enabled a more detailed analysis of the measured parameters relative to the changes in Omega-3 PhL Index. However, only weak correlations (p < 0.1) were detected when an increase in HbA1c levels or a decrease in NEFA levels was examined (see Additional file 4 http://static-content.springer.com/esm/art%3A10.1186%2Fs12986-015-0047-9/MediaObjects/12986_2015_47_MOESM4_ESM.docx). Further studies are needed to analyze the mechanisms behind the metabolic effects of interventions observed in our study, including the evaluation of changes in muscle glycogen content during the clamp. In this context, muscle glycogen was measured only in the fasting state and no differences between the subgroups were found (Additional file 1 http://static-content.springer.com/esm/art%3A10.1186%2Fs12986-015-0047-9/MediaObjects/12986_2015_47_MOESM1_ESM.docx).

In spite of the fact that pioglitazone is a well-established pharmaceutical with lasting insulin-sensitizing effects, and despite its other beneficial effects in patients with T2D, its clinical use has declined recently due to the risk of the side-effects (see Background). Importantly, at least some of these concerns have been disproved recently. Namely, it has been demonstrated that the cumulative use of pioglitazone or rosiglitazone was not associated with the incidence of bladder cancer.[39] The results of our study document beneficial effects of a relatively low dose of pioglitazone on lipid metabolism when pioglitazone was used as part of the combined intervention with n-3 fatty acids. This observation is relevant for reducing the risk of the side-effects of pioglitazone under clinical settings.

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