Short-term treatment with high dose liraglutide improves lipid and lipoprotein profile and changes hormonal mediators of lipid metabolism in obese patients with no overt type 2 diabetes mellitus: a randomized, placebo-controlled, cross-over, double-blind clinical trial

We demonstrate herein that treatment with high dose liraglutide for a short period of time (5 weeks) in obese patients without overt type 2 diabetes initiates significant alterations in lipid and lipoprotein profile compared to placebo. These alterations are partially independent of weight loss, they are similar to changes observed after long-term treatment with liraglutide in patients with type 2 diabetes and at high risk for CVD and they are indicative of a potential cardiovascular benefit [26]. Additionally, we demonstrate that hormones (i.e. follistatin and activin AB) that have been recently related to lipid metabolism, insulin resistance and glucose regulation are significantly reduced after liraglutide treatment, supporting their role as hormonal mediators of metabolic procedures in humans.

First, we observe lower levels of total, free and remnant cholesterol and apoB with liraglutide compared to placebo [1]. All these molecules are considered established markers of CVD mainly by accelerating atherogenic processes [27]. Remnant cholesterol was also reduced postprandially in patients with type 2 diabetes treated with 1.8 mg liraglutide for 16 weeks versus placebo [7]. In agreement with our findings, a recent study demonstrated significant improvement in lipid and lipoprotein profiles as a result of 14 weeks treatment with liraglutide combined with modest restriction of caloric intake [28]. Reductions in apoB have also been reported in a study including obese subjects that had initially undergone a very low-calorie diet for 8 weeks followed by treatment with liraglutide in low dose (1.2 mg) for 1 year vs. meal replacements for weight maintenance [14]. Similarly, reduced apoB48 (isoform of apoB) levels were observed after a fat-rich meal in patients with type 2 diabetes treated with 1.8 mg liraglutide [29]. The changes we observe are generally more modest compared with others, which is probably explained by the shorter duration of treatment. The mechanism involved in these changes remains unknown, albeit it has been suggested, that liraglutide may lead to reduced secretion of chylomicrons containing apoB48 and thus to decreased absorption of triglycerides [29]. In contrast to LDL, the changes we observed in HDL, VLDL and IDL were minor and do not point to a clear direction regarding their associations to cardiovascular outcome, which might be due to the relatively short duration of the study, while significant reduction of total cholesterol, LDL cholesterol, TG and non-high-density cholesterol has been demonstrated in a 14-week liraglutide treatment and associated with improvements in CVD risks and outcomes [28]. It has been suggested that liraglutide treatment might be supporting a shift from lipids to glucose oxidation, resulting in lower glucose levels and reduction in lipolysis and lipid oxidation [30].

Regarding lipid subclasses, we observed changes primarily in LDL concentrations. Firstly, the numbers of small and medium particles (S-LDL-P, M-LDL-P) were lower after liraglutide treatment compared to placebo. Number of LDL particles (LDL-P) has been positively associated with increased CVD risk and in several large studies this association was stronger than LDL-C [31‐33]. Additionally, a discordance between LDL-C levels and LDL-P or apolipoprotein B is often observed in the general population and in these cases, LDL-P and apolipoprotein B can be used to improve the assessment of CVD risk [33, 34]. Regarding particle size, small LDL particles are considered more atherogenic, since they have a longer circulation time and higher ability at penetrating the arterial wall [35]. Studies have also shown, that small LDL and cholesterol in small LDL are reliable markers of CVD [10, 36]. Thus, the lower levels of LDL-P and particularly of small LDL-P that we observe with liraglutide treatment point to a reduced CVD risk with liraglutide treatment, albeit these changes may be related to the concomitant weight loss. In a mechanistic study involving a 7-week treatment of db/db mice, liraglutide inhibited expression of proprotein convertase subtilisin/kexin type 9 (PCSK9) and low-density lipoprotein receptor (LDLR) in liver and accordingly the concentration of PCSK9 in serum and improved hepatic lipid accumulation. PCSK9 is known to play the key role in regulation of cholesterol homeostasis and is associated with glucose metabolism. Based on the mention study, PCSK9 might be one of the possible pathways through which liraglutide mediates its lipid-lowering effect [37].

Regarding the lipid content of the LDL particles, we observe a reduction in absolute concentrations of total lipids, phospholipids and free cholesterol in small LDL-P as well as in total lipids and phospholipids in medium LDL-P with liraglutide treatment and independently of weight loss. These reductions may be related to the lower number of small and medium LDL-P and consequently the lower amount of total lipids in LDL with liraglutide. When analyzed as relative concentrations to total lipids, we observe minor changes pointing towards higher free cholesterols and phospholipids and lower cholesterol esters in small and/or medium LDLs compared to placebo. Phospholipids together with free cholesterol and apolipoproteins are forming the outer hydrophilic layer of the lipoprotein which enables transportation of the hydrophobic cholesterol esters and triglycerides located in the inner part. Whether longer duration of liraglutide treatment affects the relative lipid content of LDLs and consequently their structure and metabolism have not been investigated yet.

Regarding lipid classes and fatty acids, sphingomyelins were lower with liraglutide treatment compared to placebo independently of weight loss. Interestingly, levels of two individual sphingomyelin species have been previously positively associated with CVD risk in a large prospective study (Bruneck study) [38]. Our study design did not allow us to identify which specific sphingomyelin species are reduced with liraglutide. In contrast, we did not observe any changes in phosphatidylcholines, phosphatidylglycerols or total cholines. Regarding fatty acids, slightly lower concentrations of PUFA, omega-3 and omega-6 fatty acids were observed, which were partially weight-loss related. An interesting effect of liraglutide treatment was the raise of behenic acid (C22:0), which lasted even after adjusting for weight loss. There is increasing epidemiologic evidence on the cardiometabolic benefits of increasing circulating concentrations of this very-long chain saturated fatty acid, the status of which is marginally influenced by its dietary intake. Specifically, higher levels of behenic acid are associated with a lower risk for diabetes, even after adjusting for diabetes risk factors and adiposity [39] as well as with lower risk of incident heart failure [40] and lower incidence of coronary heart disease (CHD) [41].

Studies of liraglutide and metformin combination have shown that liraglutide treatment might be more effective for reduction in non-estherified fatty acids (NEFA) and LDL profile in patients with newly diagnosed type 2 diabetes compared with metformin due to its ability to enhance insulin secretion and inhibit inflammatory cytokines [30, 42].

We also observe a reduction in circulating tyrosine levels with liraglutide. Tyrosine is a non-essential amino acid that is involved in signal transduction mechanisms. Phosphorylation of tyrosine through tyrosine kinases and dephosphorylation by protein tyrosine phosphatases are part of signaling pathways that are implicated in fundamental cellular functions, including cell growth, differentiation and oncogenic transformation [43]. Significant elevation of tyrosine has been identified in individuals with metabolic disorders [44] and had highly significant associations with future diabetes [45]. Tyrosine kinase inhibitors used for treatment of malignancies have demonstrated improvement of glycaemia in cases of type 1 diabetes and type 2 diabetes, possibly by enhancing beta cell survival and insulin secretion [46]. However, a clear link between circulating tyrosine levels and insulin resistance, diabetes or CVD risk has not been established yet.

Finally, we observe a significant reduction in follistatin and in activin AB. Follistatin is mainly secreted by the liver and its inactivation in hyperglycemic mice improves glucose homeostasis by reducing insulin resistance [18]. We have recently demonstrated that glucose or the concomitant insulin release during OGTT reduces follistatin levels, suggesting the presence of a feedback loop mechanism controlling the effects of follistatin on glucose metabolism [16]. In line with these findings, we have also reported a reduction of follistatin in morbidly obese patients early after bariatric surgery, which predicts the improvement in insulin sensitivity observed later in these patients. In this study, we expand our findings by showing that short term treatment with liraglutide reduces robustly circulating follistatin. This reduction is weight-independent but HOMA-IR dependent, confirming the link of follistatin with insulin sensitivity. Follistatin is also the natural inhibitor of activins. Although, activin A and B do not change with liraglutide treatment, a robust decrease of activin AB is observed. Activin AB is a heterodimer of inhibin βA and inhibin βB, which as dimers form activin A and activin B respectively [47]. Thus, the explicit reduction in activin AB and not in A or B indicates the involvement of mechanisms downstream of gene expression and probably in mechanisms defining heterodimer assembly and secretion. In contrast to insulin sensitivity, both the absolute as well as the delta changes of follistatin and activin AB were not associated with the changes observed in lipid–lipoprotein profile arguing against a significant involvement of these hormones in lipid metabolism. Future mechanistic studies should aim to investigate whether a direct GLP-1/Follistatin–Activin AB crosstalk exists that contributes to the regulation of glucose homeostasis.

The current study has some strengths and limitations. A major strength is the double-blinded placebo controlled cross-over design which reduces potential confounders and increases power. Additionally, the measurements were performed from personnel blinded for treatment groups. A limitation is the lack of postprandial blood draws, which would have been important mainly for parameters participating in the exogenous pathway of lipid metabolism (i.e. chylomicrons). Finally, our study was focused on lipoproteins and large lipid classes and did not include an untargeted analysis of individual lipid species.