Animals on a HFD develop hyperglycemia in conjunction with hyperlipidemia, indicating the manifestation of insulin resistance. The improved glycemia, fasting serum insulin and oral glucose tolerance observed in response to metformin treatment was expected and in line with the known anti-diabetic properties of metformin [
16,
17]. The oral gavage administration did cause nausea and reduced food intake during the 14 days of treatment, where those mice receiving a HFD displayed significantly reduced appetite. Further, metformin treatment initially induced diarrhea. Starting with a lower dosage and increasing up to desired dosage over 3–4 days may have avoided this side-effect of metformin treatment. To detect if the weight loss during treatment was a confounding factor for the beneficial effects of metformin, we evaluated weight as an independent parameter for the improved metabolic state. The fact that weight alone displayed a significant positive correlation with HbA
1c, but not fasting serum insulin, indicates that -- although the weight loss most likely contributed to the beneficial effects -- metformin treatment was indeed effective. In contrast to numerous reports of enhanced GLP-1 secretion in response to dietary fat [
7,
8], no significant increase in fasting or prandial GLP-1 (7–36 and/or 9–36) could be detected in our animals fed a HFD. This discrepancy may stem from differences in the fatty acid stimuli; lipid load/HFD, as well as the duration of the HFD. In fact, many of the before mentioned studies investigating effects of fatty acids on GLP-1 secretion have focused on acute effects, and it can be hypothesized that toxic effects of chronic hyperlipidemia and reduced L-cell mass at some stage outweigh the stimulatory effect of the enteral fatty acids on GLP-1 secretion,
i.e. exceeding the ability of the remaining L-cells to compensate. Extending the duration of the HFD may thus eventually result in a defective postprandial GLP-1 response, while reducing the duration of the HFD may result in observations of increased plasma GLP-1 in this group, as was indeed recently demonstrated [
18]. This hypothesis is supported by the trend toward reduced numbers of GLP-1-positive cells detected in the intestinal tissue from HFD-fed mice as compared to intestinal tissue from mice receiving a control diet, and in line with reports of a negative correlation between GLP-1 plasma levels and BMI [
12]. Further, metformin was indicated to counteract such effects of a HFD on the number of GLP-1-positive cells. Detrimental effects only after persistent and long term exposure to hyperlipidemia could theoretically be explained by an accumulation of FFAs that eventually exceeds the capacity of the L-cell for triglyceride storage and the subsequent increase in β-oxidation and ROS production [
10,
13]. However, measurement of plasma GLP-1 in the different treatment groups following a glucose load would have added to the understanding of potential effects of a high fat diet and metformin treatment on the incretin response, and should be undertaken in future studies.
The present study shows reduced intestinal proglucagon expression in mice on a HFD following metformin treatment, which aligns well with previously published in vitro data, indicating metformin stimulatory action to be at the level of secretion. However, if metformin treatment increases the number of viable GLP-1-positive cells after a HFD, an increased intestinal proglucagon expression would be expected despite possible counteracting effects of metformin on proglucagon expression at the level of individual L-cells. The interpretation of the available data from this study is made even more difficult due to the adverse gastrointestinal side-effects induced by the metformin treatment, and the relatively short duration of metformin treatment. Therefore, further studies are necessary to confirm whether metformin lipoprotection can be observed in vivo, or is purely an in vitro phenomenon. The upregulation of GLP-1R mRNA in response to a HFD -- and normalization thereof by metformin treatment -- provokes further assessment of the intestinal expression of the GLP-1R under these conditions. Further, the mechanisms behind such effects remain to be investigated. It is, in light of a defective incretin response in diabetic patients improved by metformin treatment, tempting to hypothesize that compensatory mechanisms underlie increased receptor expression in response to reduced levels of the ligand and/or defective GLP-1R signaling in HFD-induced T2D.
In conclusion, our findings provide evidence for rapid development of insulin resistance and diabetes in mice receiving a HFD, with a significant improvement in response to metformin, which also was indicated to improve the prandial incretin response of HFD-fed mice. Further, our data demonstrate a clear trend toward reduced numbers of GLP-1-positive cells in HFD mice. However, considering no significant effect on fasting or prandial levels of GLP-1 in response to HFD, any reduction in the number of GLP-1-positive cells seemingly does not contribute to the development of oral glucose intolerance, hyperinsulinemia and hyperglycemia in this study. It may rather contribute to the progression of the diabetic state as it may lead to decreased prandial GLP-1 secretion when the reduction of GLP-1-positive cell number in response to hyperlipidemia overtakes fatty acid-induced potentiation of GLP-1 secretion.