Influence of sphingolipid enzymes on blood glucose levels, development of diabetes, and involvement of pericytes

Sulfatide is a chaperone for insulin manufacturing in beta cells. Here we explore whether the blood glucose values normally could be associated with this sphingolipid and especially two of its building enzymes CERS2 and CERS6. Both T1D and T2D have low blood sulfatide levels, and insulin resistance on beta cells at clinical diagnosis. Furthermore, we examined islet pericytes for sulfatide, and beta‐cell receptors for GLP‐1, both of which are related to the insulin production.


| INTRODUCTION
Sphingolipids are a distinctive class of lipid molecules discovered over a hundred years ago and named after the sphinx due to their mystic nature.They consist of two fatty chains, an amino acid (serine), and a sugar fragment.Their building ways are well known from the uptake of serine to the following biochemical reactions catalysed by specific enzymes. 1 Two of the important enzymes are CERS2 and CERS6, which convert dihydrosphingosine to dihydroceramide.Notably, CERS2 performs longer isoforms, while CERS6 performs shorter fatty chain variants. 2The role of sulfatide (sulphated beta galactosyl ceramide) has been studied extensively in the pancreatic beta cells in the islets of Langerhans. 3 Sulfatide acts as a chaperone for insulin by facilitating folding of insulin, preserving its crystallisation at a low pH, and aiding in the exocytosis of the secretory granules.Following insulin secretion, sulfatide provides negative feedback to the individual beta cells, preventing further insulin secretion until new secretory granules have accumulated near the cell membrane.Overall, sulfatide inhibits beta-cell destruction and reduces the toxic effect of cytokines. 4Interestingly, the physiological actions seem mediated by the C16 chain length, while the immunological functions are associated with the C24 isoforms. 5though Type 1 Diabetes (T1D) and Type 2 Diabetes (T2D) have different etiopathogenesis, there are shared elements such as betacell stress and influence of cytokines. 6,7Both T1D 1 as well as T2D 8 exhibit diminished levels of sulfatide.During development of prediabetes, impaired glucose tolerance will take place at a certain point.This will occur together with relatively hyperinsulinism and insulin resistance. 9At T1D diagnosis, approximately one-third of the beta cells are inactive.The reason for the inactivity of the beta-cells could partly be due to the low number of insulin receptors, which indeed is the case. 10,11Also, the incretin hormones GLP-1 and GIP are important for the insulin secretion, and their receptors at the beta cells are studied here.
Additionally, during the prediabetic stage of T1D, the capillaries within the islets are affected: they enlarge and decrease in number, 12 while their pericytes become less functional. 13Normally, postprandial when blood glucose values increase, capillaries open to provide adequate energy for the surrounding beta cells to produce the relevant amount of insulin.
In the present study, we examined incretin receptor mRNA levels in pancreatic islets obtained from living, newly diagnosed T1D patients from the Norwegian DiViD study 14 and pancreatic islets from healthy organ donors or with T1D or T2D from the American nPOD study. 15Furthermore, we have analysed polymorphisms related to blood glucose levels and HbA1c for the above-mentioned sphingolipid enzymes CERS2 and CERS6.Finally, we examined normal islets for the presence of sulfatide in their pericytes.

| RNA analysis
Human pancreatic tissue utilised in this study was obtained from the DiViD study 14  (reference 10-00848-XM).Direct to lars.krogvold@gmail.comfor requests to the datasets.As opposed to the living patients in the DiViD study, the nPOD study consists of pancreases from organ donors following accidental death.Frozen sections of the pancreatic tissue were subjected to laser capture dissection 16 and islets from two to five sections were pooled, and RNA was extracted, using the Arcturus PicoPure RNA Isolation Kit (Applied Biosystems, Grand Island).RNA quantification was performed using a Bioanalyser 2100 instrument (Agilent Technologies).Gene expression analysis was conducted using Affymetrix expression arrays (GeneChip Human Gene 2.0 ST, Thermo Fisher) and normalised using global scaling. 17 ensure consistency in the comparison, all tissue handling procedures were carried out in the same laboratory, by the same technicians.Furthermore, the analysis was performed using the same equipment, and the RNA quality was certified by RIN value measurements (>3.5).

| Light microscopy
Immunohistochemistry was performed on representative formalinfixed paraffin-embedded whole sections 3μm thick.Sections of the pancreas were obtained from 10 non-diabetic persons examined at the Department of Pathology, Rigshospitalet.As we are a University Hospital, it is allowed for our pathological department to use specimens anonymously for scientific purposes.For the staining protocol, sections were deparaffinised in EZ prep (#950-102, ready to use, Ventana Medical Systems) and HIER was performed with CC1-buffer (#950-124, ready to use, Ventana Medical Systems).The sections were double stained with a primary antibody Sulph I for sulfatide as described previously in 25

| Statistical analysis
Statistical analysis for RNA expression was conducted using Graph-Pad Prism 8.0.2 (GraphPad, La Jolla).Outliers within each group were identified using the ROUT method and a total of 21 data points across all genes and groups were identified and removed.All groups were tested for normal distribution using the D'Agostino-Pearson and Anderson Darling tests.For comparison between groups, a oneway ANOVA was employed followed by Dunnett's multiple comparison test and a 95% CI.Statistical significance was considered when p < 0.05.

| RESULTS
We identified a significant association with T2D for polymorphisms in both CERS2 (p = 7 � 10-E7) and CERS6 (p = 1 � 10-E9) genes (Table 1).For T1D patients, no associations were observed, but the CERS2 enzyme mRNA level was earlier found to be depressed to 26% in T1D. 1 In non-diabetic persons, highly significant polymorphisms in CERS6 were linked to random blood glucose levels, whereas polymorphisms in CERS2 were associated with HbA1c (Table 1).Given that CERS2 is a precursor enzyme for sphingolipids, this aligns well with our discovery of sulfatide in pericytes in the islets of all ten examined non-diabetic persons, which regulates the capillary blood flow in the islets of Langerhans, important for oxygen supply to insulin production (Figure 1).
During late development of T1D, dysfunctional pericytes are observed in the islet together with larger but fewer capillaries leading to reduced oxygen supply and impacting the insulin production.This latter can also be worsened otherwise.We examined the receptors of the incretin hormones on islet cells.Regarding the GLP-1 receptor, the newly T1D patients display a reduction in its expression, being 46.6% of the value in healthy controls (p = 7 � 10-E8) and 36.0% of the value for the antibody-positive persons (p = 3 � 10-E8).Additionally, the GIP receptor mRNA expression was diminished in islets from new T1D patients: 79.0% compared with control persons (p = 0.04).Since low GLP-1 receptor function compromises insulin secretion, this finding adds to our understanding of the T1D pathogenesis.

| DISCUSSION
Sphingolipids are biological intermediates in glucose metabolism 26 and polymorphisms in genes central in the sphingolipid pathway are associated with glucose metabolism, that is, blood glucose levels and HbA1c.This robust connection is interesting considering a study 13 in which it is found that pericytes in the islets should allow increased capillary blood flow as the blood glucose levels rise to facilitate increased oxygen supply to beta cells having high(er) activity of insulin production.In the present study, we found sulfatide in the pericytes of islets, as shown in Figure 1.The CERS6 enzyme is related to C16:sulfatide (short fatty chain length), whereas CERS2 performs the C24:sulfatide isoform.Interestingly, the C16:sulfatide is associated with physiological insulin secretion, which is not the case for the C24 isoform 3 , suggesting that the HbA1c polymorphism related to the C24 isoform might be mediated by a pericyte mechanism rather than via beta cells.
Beta cells are often highly metabolically active, and it is crucial that the oxygen supply is optimal and thereby a sufficient blood flow.
This has been found to decrease prior to clinical T1D in both animal models and humans. 12In the pre-diabetic states in the islets, capillaries are dilated but in smaller numbers, resulting in diminished oxygen supply for many beta cells 12 which then are stressed and hence more sensitive to immune attacks. 27ere appears to be a parallel to diabetic retinopathy, which is caused by the sensation of hypoxia in the retina.We previously showed that pericytes in the choroid layer contain plenty of sulfatide molecules, 28 and during retinopathy they are in poor shape or are disappearing. 29The role of sulfatide in this context is unknown but a suggestion might be that it serves as a reservoir anions (by the negative sulphate group in sulfatide) prepared for Ca þþ used for the smooth muscles in the pericytes.Notably, flushing in the face after alcohol intake can be demonstrated in one-third of patients with T2D 30 , which might be caused by opening of capillaries, which again means presence of active pericytes.Interestingly, in patients with T2D, less retinopathy is found among those exhibiting the flushing reaction. 30Furthermore, fenofibrate treatment increases the amount of C24:sulfatide, and this treatment is beneficial against retinopathy in humans. 31 the islets of newly diagnosed T1D patients, poorly functioning capillaries have been described. 12At diagnosis of T1D this might be due to the known significant decrease in the amount of sulfatide in the islets, which we have found to be only 23% of the normal values. 1 Although not entirely proven, it seems that the amount of sulfatide is crucial for beta cells both regarding the physiological function 3 and for their energy consumption mediated by relevant blood flow.
Also, in T2D, a low amount of sulfatide seems relevant.This is supported by the Swedish Skaraborg study where we found an association of double risk to have T2D when sulfatide levels were in the lowest or middle tertiles.Apart from poorly functioning pericytes, low insulin secretion during the development of the T1D disease may be due to another parallel mechanism related to beta-cell insulin and incretin receptor expression levels.At the time of T1D diagnosis, roughly onethird of the beta cells have been destroyed, one-third are infiltrated by immune cells, mainly lymphocytes, and one-third are 'sleeping'; these appear microscopically normal but produce no insulin and are not infiltrated. 14We have found among newly diagnosed T1D patients, that the insulin receptors were 53.4% of the value for the control persons (p = 0.00003). 11In contrast to the study of Kahn, 10 a recent study using living animals showed that lack of insulin receptors on the beta cells increased the insulin production and secretion. 32e beta-cell mass adapts to a lower activity level, for example, after a pregnancy or other situations, when the insulin need is reduced. 33On the other hand, lizards (Glia Monster) that eat only every other week, preserve their beta cells between meals.This is also observed for bears during hibernation.A difference between these conditions is the level of GLP-1, suggesting that this hormone is necessary for mediating reduction of inactive beta cells.Thus, the lizard and the bears do not produce GLP-1 during their inactivity, as is the case after pregnancy, where women eat normally.This led us to hypothesise that a low level of GLP-1 receptors -as found in the present study -might be crucial for beta-cell preservation likely in combination with islet insulin resistance.
During the immune attack on the beta cells leading to the diagnosis of T1D, it is beneficial for the individual beta cells to be less metabolically active, which in this article is referred to as 'sleeping', to avoid destruction.During relaxation, less quantity of self-antigens is exposed 27 ; there is less sensitivity to cytokines 34 ; and overall, the beta cells are less vulnerable.On the other hand, this sleepiness is not solidaric with the few active beta cells that produce all the needed insulin even though they are under immune attack, and therefore become more vulnerable.
The present findings strengthen the connection between diabetes, pericytes, sulfatide, and receptors of incretin hormones on beta cells.

F I G U R E 1
Pancreatic sections from non-diabetic human persons stained with anti-pericytes (purple) and Sulph I against sulfatide (teal).Co-localization is seen at several spots in the islet structures.(A), (B) and (C) are from the same person in magnification 1:10, 1:20 and 1:40, respectively.(D)-(L) are pancreatic sections from 9 non-diabetic persons in magnification 1:20.
Table showing the various SNPs and genes with polymorphisms in this study.