A High-Fat Diet Increases Activation of The Glucagon-Like Peptide-1-Producing Neurons in The Nucleus Tractus Solitarii; An Effect That is Partially Reversed By Drugs Normalizing Glycaemia

Glucagon-like peptide-1 (GLP-1) is a peripheral incretin and centrally active peptide produced in the intestine and nucleus tractus solitarii (NTS), respectively. GLP-1 not only regulates metabolism but also improves cognition and is neuroprotective. While intestinal GLP-1-producing cells have been well characterized, less is known about GLP-1-producing neurons in NTS. We hypothesized that obesity-induced type 2 diabetes (T2D) impairs the function of NTS GLP-1-producing neurons and glycaemia normalization counteracts this effect. We used immunohistochemistry/quantitative microscopy to investigate the number, potential atrophy, and activation (c-Fos-expression based) of NTS GLP-1-producing neurons, in non-diabetic versus obese/T2D mice (after 12 months of high-fat diet). NTS neuroinammation was also assessed. The same parameters were quantied in obese/T2D mice treated from month 9 to 12 with two unrelated anti-hyperglycemic drugs: the dipeptidyl peptidase-4 inhibitor linagliptin and the sulfonylurea glimepiride. We show no effect of T2D on the number and volume but increased activation of NTS GLP-1-producing neurons. This effect was partially normalized by both anti-diabetic treatments, concurrent with decreased neuroinammation. Increased activation of NTS GLP-1-producing neurons could represent an aberrant metabolic demand in T2D/obesity, attenuated by glycaemia normalization. Whether this effect represents a pathophysiological process preceding GLP-1 signaling impairment in the CNS, remains to be investigated.

reported that NTS-produced GLP-1 can speci cally modulate a satiation/satiety circuit under stress conditions and after large meals (Holt et al. 2019). Consequently, there is an increased need to better understand the role of these cells under physiological and pathophysiological conditions such as T2D.
The aim of this study was to compare the effects of a standard "healthy" diet versus an "unhealthy" obesogenic diet inducing T2D, on the function of GLP-1-producing neurons in NTS. The used obesogenic diet included high-fat content but also differed from the standard diet in terms of composition and amount of energy coming from other energy sources. Speci cally, we investigated whether this obesogenic diet inducing-T2D could affect the number, induce atrophy and/or induce changes in neuronal activation of GLP-1 producing neurons. We also investigated whether sustained glycemia normalization could counteract such effects.

Animal model and experimental design
In accordance with the guidelines aiming to improve the ethical use of animals in experimental research (3R principle) (Balls 2009), the material (brain and plasma) came from the same mice cohort as recently used for another study. Therefore, part of the metabolic data has already been published (Lietzau et al. 2020).
Twenty-ve, male C57Bl/6J mice (Charles River, Germany) were housed under controlled conditions, with ad libitum access to food and water. All applicable international, national, and institutional guidelines for the care/use of animals were followed. All experimental procedures were in accordance with the ethical standards of the Karolinska Institutet (ethical approval no. S7-13).
Two-months-old mice were randomly assigned to 4 experimental groups. The potential effect of obesityinduced T2D on GLP-1-producing neurons and neuroin ammation in the NTS was determined by comparing outcome parameters in mice fed an obesogenic diet enriched in fat (high-fat diet (HFD): 54% calories from fat, ssniff® E15126-34, Germany) (n = 6) for 12 months with age-matched mice fed a standard diet (ENVIGO 2018, Italy; SD) (n = 7). The potential effect of sustained glycemia normalization was determined by adding two groups: HFD-fed mice administered (in food) with either linagliptin (average dose of 5-7 mg/kg b.w. per day; HFD-Lina) (n = 6) or glimepiride (average dose of 2-4 mg/kg b.w. per day; HFD-Gli) (n = 6) for 3 months before sacri ce (between month 9 and 12). Both drugs are clinically prescribed for the treatment of T2D, but they have different mechanisms of action. Linagliptin is a dipeptidyl peptidase-4 inhibitor (DPP-4i) that prevents degradation of endogenous GLP-1, which results in increased insulin secretion/sensitivity (Deacon and Holst 2013). Additionally, DPP-4i also have neuroprotective action (reviewed in (Darsalia et al. 2019) and (Chalichem et al. 2017). Sulfonylureas, such as glimepiride, induce direct pancreatic insulin secretion (Khunti et al. 2018). We hypothesized that an effect induced in NTS by both drugs would suggest that it is related with glycemia regulation, but induced only by linagliptin would suggest another, glycemia-independent mechanism. Body weight, glycaemia, DPP-4i activity and GLP-1 levels Body weight and fasting blood glucose were measured in all mice. Plasma DPP-4 enzyme activity and total active GLP-1 levels were determined (fed state) by EIA and ELISA, respectively (MesoScale Discovery, USA).

Immunohistochemistry and quantitative microscopy
An immuno uorescence staining protocol to quantify GLP-1-producing neurons was applied (Supplementary le) using a NewCast system (Visiopharm, Denmark), connected to Olympus BXS51 microscope (Olympus, Japan). Activation of GLP-1-producing neurons was assessed by quantifying GLP-1/cFos + cells (Rinaman 1999). To evaluate potential atrophy, mean volume of GLP-1/cFos + cells was measured using the nucleator technique (Gundersen et al. 1988). Potential activation of neuroin ammation in the NTS was quanti ed as density/mean volume of Iba-1 + microglia cells and total number of CD68 + microglia cells. Morphological analyses were performed by a blinded experimenter.

Statistical analysis
Data were checked by the Shapiro-Wilk normality test. All studied parameters were analyzed as follows: 1. SD versus HFD group comparisons were performed using unpaired, two-tailed t-test with Welch's correction. 2. HFD versus HFD-Lina versus HFD-Gli comparisons were performed using ordinary one-way ANOVA followed by uncorrected Fisher's LSD test.
Data are expressed as mean ± SD. p < 0.05 were considered statistically signi cant. All data were analyzed by GraphPad Prism 8 (USA).

Results
Twelve months of HFD intake increased body weight compared to SD-fed mice (p = 0.0055) (Fig. 1A).
Neither 3-month-treatment with linagliptin nor glimepiride had any effect on the body weight (Fig. 1A). HFD-feeding also resulted in fasting hyperglycemia (p < 0.0001 compared to the SD), normalized by both anti-diabetic drugs (p < 0.0001 compared to HFD-Lina and HFD-Gli) (Fig. 1B). In linagliptin-treated mice, the plasma activity of DPP-4 enzyme was, as expected, decreased compared to both the HFD and HFDglimepiride groups (p < 0.0001 for both comparisons) (Fig. 1C). Furthermore, linagliptin-treated mice showed a higher concentration of active GLP-1 compared to HFD (p < 0.0157) and a similar trend, although not statistically signi cant (p = 0.1234), was observed in comparison to the HFD-Gli group (Fig. 1D). Neither difference in DPP-4 enzymatic activity nor GLP-1 plasma concentration was detected between the HFD and HFD-Gli mice (p = 0.2431 and p = 0.5973, respectively) ( Fig. 1C-D).
No difference was detected in the total number of NTS GLP-1-producing neurons between HFD-and SDfed mice, suggesting no change in GLP-1 expression or cell death. Additionally, no effect of linagliptin or glimepiride was recorded ( Fig. 2A-B). However, we observed an increased number of NTS cFos + cells in the HFD compared to SD group (p = 0.0008), suggesting an increased cellular activation in NTS, with no additional effects of the anti-diabetic drugs (Fig. 2C-D). The number of double-stained GLP-1/cFos + cells was increased in the HFD group (p < 0.0001 compared to the SD group) (Fig. 2E-F) indicating increased activation of GLP-1 + cells. Both linagliptin (p = 0.0426) and glimepiride (p = 0.0158), partially, but signi cantly, counteracted this effect (Fig. 2F). To examine if these effects occurred speci cally in GLP-1producing neurons, we subtracted the fraction of cFos/GLP-1 + neurons from the total number of NTS cFos+/activated cells. The effects of HFD, linagliptin and glimepiride were lost after this subtraction ( Fig. 2G), indicating that the recorded increase/decrease in the number of NTS cFos + cells (and, thus, indirectly in their activation) induced by HFD and anti-hyperglycemic treatments, respectively, occurs speci cally in GLP-1-producing neurons.
Twelve months of HFD-feeding had no effect on the mean volume of activated GLP-1/cFos + cells (Fig. 2H). However, we recorded a signi cant decrease in this parameter in the HFD-Gli group (p = 0.0215 compared to HFD, and p = 0.0037 compared to HFD-Lina), suggesting increased cell atrophy.
No signi cant difference in Iba + cells density in NTS between SD-and HFD-fed mice ( Fig. 3A and C), or in response to anti-hyperglycemic drugs was detected (Fig. 3B-C).Interestingly, both linagliptin and glimepiride signi cantly reduced the volume of NTS Iba + cells in HFD-fed mice (p = 0.0252 and p = 0.0449, respectively) ( Fig. 3A-B and D), suggesting decreased neuroin ammation. We observed no CD68 + cells in any of the studied groups (data not shown).

Discussion
We hypothesized that obesity-induced T2D obtained after neurons may indicate that this effect occurs via glycemia regulation, since these drugs normalize glycaemia through unrelated mechanisms.
No difference in neuroin ammation between SD-and HFD-fed mice was detected after 12 months in NTS, which may indicate lack of an association between changes in the basal activity of GLP-1producing neurons induced by HFD and increased neuroin ammation Though speculative, HFD may increase neuroin ammation at earlier time points, as previously reported (Speretta et al. 2019;Butler et al. 2020). This effect could be masked at 12 months by increased neuroin ammation during the normal aging process (Cribbs et al. 2012). A decreased volume of Iba + cells was recorded in response to linagliptin and glimepiride suggesting decreased T2D-induced NTS neuroin ammation after 12 weeks of glycaemia normalization. Future studies will have to demonstrate whether decreased NTS neuroin ammation of obese/T2D mice after glycaemia normalization could represent a causal factor or a consequence of the normalization of the T2D-induced activation of NTS GLP-1-producing neurons.
The preliminary ndings of this short communication need to be con rmed using direct methods to assess NTS neuronal activation. Additionally, electrophysiological studies should be performed to investigate whether the changes in the activation of NTS GLP-1-producing neurons are re ected in altered GLP-1R activation / GLP-1 content in areas targeted by these cells.
In conclusion, our study provides new insights into the effects of obesity-induced T2D on NTS GLP-1producing neurons that could lead to development of new strategies to improve GLP-1 mediated metabolic control and neuronal function in the CNS. Code availability NA Authors' Contributions GL coordinated and performed IHC studies and stereology analyses, acquired and processed images/ gures, contributed to discussion, wrote, and edited the manuscript. SN, HP, TC performed IHC studies, analyzed the data and edited the manuscript. VD performed in vivo studies and edited the manuscript. TK provided linagliptin and glimepiride, and edited the manuscript. LT contributed to discussion and edited the manuscript. TN provided resources, contributed to discussion, and edited the manuscript. CP and CK -conceived, designed and coordinated the research plan, contributed to discussion, and wrote the manuscript.