In this study, we show that the acute effects of NEFA on insulin secretion are asymmetric. While in non-diabetic volunteers NEFA elevation caused a marked impairment of incretin-induced potentiation, in individuals with type 2 diabetes the impaired incretin effect was not improved by acutely lowering plasma NEFA. Of note, in the non-diabetic participants raising NEFA induced all the expected metabolic changes [9, 22]: mild deterioration of glucose tolerance and insulin sensitivity, increased insulin secretion and enhanced early insulin response (i.e. rate sensitivity) without any changes in β-GS. Conversely, acipimox administration in diabetic participants was associated with reduced insulin secretion and plasma glucose excursions and improved insulin sensitivity, but neither beta cell glucose sensitivity nor incretin-induced potentiation was changed. Of interest is that the effects of plasma NEFA modulation were observed in both the oral and i.v. tests; this rules out a role for the entero–insular axis or the taste receptors/cerebral–insular axis. In support of this, neither lipid infusion nor acipimox was associated with any changes in plasma glucagon, GLP-1 or GIP responses in the participants with or without diabetes. The absence of short-term changes in gut hormones agrees with studies in normal volunteers subjected to 1 week of high-fat overeating, in whom glucose tolerance deteriorated, plasma insulin increased but GLP-1 and GIP responses were not modified .
Taken together, these findings make it possible to conclude that the direction of the impact of NEFA on incretin effect depends, essentially, on beta cell function: raising NEFA can impair the incretin effect on a normal beta cell but lowering NEFA cannot rescue the incretin effect on a dysfunctional beta cell. In line with the latter result, lipid-induced impairment of beta cell function is not observed in frankly diabetic animals or humans . Obviously, our conclusions apply only to acute changes in NEFA concentration and to the experimental conditions created by these protocols, i.e. they provide proof of concept. In the longer term, the impact of NEFA on beta cell function may be more heterogeneous. For example, Kashyap et al  reported that in normal participants a prolonged (4 day) lipid infusion led to mild meal-induced hyperglycaemia and insulin hypersecretion, which was associated with a marked improvement in first-phase insulin secretion. In contrast, opposite responses were observed in individuals with a family history of diabetes . However, in a similar study, 48 h acipimox treatment improved the acute insulin response to i.v. glucose in individuals with a family history of type 2 diabetes . It must be observed, however, that these studies did not directly assess the incretin effect, which, by definition, requires performing an isoglycaemic protocol. On the other hand, it is pertinent to recall that chronic treatment with a dipeptidyl peptidase IV (DPP-IV) inhibitor, sitagliptin  or metformin , in diabetic individuals did not improve the incretin effect.
The current results align with those of a previous study carried out in a large cohort of non-diabetic individuals , which indicated that relatively higher NEFA levels, such as are typically found in type 2 diabetic individuals, are associated with enhanced absolute ISRs and reduced insulin action, but they do not interfere with β-GS, which is the strongest determinant of glucose tolerance.
Several cellular mechanisms may be involved in the impairment of incretin function caused by NEFA exposure. Kang et al reported that, in rat insulinoma INS-1E cells and in isolated islets of db/db mice, palmitate decreases the expression and levels of the GLP-1 receptor; this was associated to impairment of: cAMP production, protein phosphorylation of cAMP-responsive elements binding protein (p-CREB) and insulin secretion . A recent very elegant study investigated the effect of palmitate on the recruitment, by GLP-1, of the beta cell network, which is essential to synchronise rapid increases in glucose-induced insulin secretion. Gap junctions in human beta cells, through connexin 36 (Cx36), are important for incretin-stimulated but not for GSIS . In these human islet preparations, palmitate reduced Cx36 expression and the coordinated secretory activity in response to GLP-1 and GIP, thereby reducing insulin release. Thus, elevated NEFA might disrupt the GLP-1-sensitive syncytium. Chronic mechanisms may involve accumulation of malonyl-coenzyme A (CoA) and long-chain fatty acyl-CoAs, increased fatty acid esterification and activation of endoplasmic reticulum stress in beta cells (reviewed in Giacca et al  and Poitout and Robertson ). Decreased free fatty acid receptor 1 (GPR40) expression in the beta cell, or its downregulation by NEFA, may contribute to the impaired incretin action observed in diabetes. It is noteworthy that our diabetic participants had higher fasting glucose and marginally higher fasting NEFA concentrations (700  vs 588 , p = 0.12), a combination that seems to be synergistically toxic to islets , where it downregulates GPR40 expression in all cell types . Similar insulin secretion impairment was observed in human islets exposed to palmitate or oleate for 48 h . GPR40 is highly expressed in islets from young normoglycaemic, hyperlipidaemic and prediabetic Zucker diabetic fatty (ZDF) rats (fa/fa) even before diabetes development . Furthermore, in rats maintained under hyperglycaemic conditions for 96 h, GLP-1 receptor expression is reduced, and GIP receptor expression is increased . While these mechanisms, alone or in combination, may underlie the effect of raising NEFA into the millimolar range we observed in our non-diabetic participants, a role for the mild but chronic NEFA elevations, commonly found in individuals with type 2 diabetes, to the loss of incretin effect remains to be conclusively demonstrated. The absence of changes in gut hormones across our NEFA manipulations implies that an impaired incretin effect—whether induced in non-diabetic participants or spontaneous in diabetes—is coupled with a degree of cellular resistance to incretins . Interestingly, in a recent study in type 2 diabetic individuals, the clinical efficacy of liraglutide, a GLP-1 receptor agonist, was related to the baseline plasma triacylglycerols and C-peptide levels .
Limitations and strengths
These studies were carried out in two centres and the general phenotypes of the two participant groups were different. We chose a very healthy group to test the effects of NEFA, and typical individuals with type 2 diabetes to evaluate a possible improvement in incretin effect after NEFA reduction. The results were not meant to be compared across groups but within groups. Another limitation is the sample size, but the incretin effect, i.e. the main outcome variable of the protocol, was very different after lipid infusion in the non-diabetic participants, and very similar after acipimox ingestion in the diabetic participants. On the other hand, this is the first study to evaluate the effect of NEFA on the incretin effect in humans with an appropriate protocol. The mathematical model yielded the dynamic characteristics of beta cell function, including the time course of glucose potentiation and the incretin potentiation of insulin secretion.