Two Weeks High Glucose is Enough to Induce Liver and Gut Lesion

Background: High glucose is critical for diabetes.But in which way it induces diabetes, and which organ trigger the formation of diabetes are not clear.This study is to evaluate the effect of shot time high glucose on different organs,to make clear this question. Methods: Twelve weeks old SD rats were randomly assigned to control group,high glucose infusion (HGI) group and oral high glucose (OHG) group.Fasten blood sugar,TNF-a and IL-6 were measured.Kidneys,intestine and liver samples were collected for pathological examination.Feces of rats were collected for gut microbiota tests. Results: The results indicated that short time high glucose induced hyperglycemia lasted for at least 2 weeks after ceasing of high glucose.It increased serum levels of IL-6 and TNF-a obviously.It led to small intestinal mucosa injury, obvious steatosis of hepatocytes, and broke the balance of gut microbiota.OHG led to swelling and necrosis of individual intestinal villi.HGI led to necrosis and disappearence of cells in the upper layer of intestinal mucosa.The lesion was conned to the mucosa.There is not obvious biopsy change in kidney and pancreas. Conclusions: Short time high glucose induced lesion of liver and intestine,broken the balance of gut microbiota.All of these led to inammation and triggered diabetes.


Introduction
Type 2 Diabetes mellitus(T2DM) is rapidly increasing worldwide. It is linked to higher medical costs [1,2].
Diabetes treatments have made some success.But their effect is not always sustained and their use may associated with undesirable side effects, such as hypoglycaemia. Insu cient blood glucose control led to complications and early mortality.The complications of diabetes ,like cardiovascular disease, nephropathy, neuropathy, liver damage and so on happen quickly after formation of T2DM [3].Some clinical experiments con rmed the "legacy effect" or "metabolic memory" of prior glycemic control.Longterm cardiovascular bene ts of good glycemic control early in the course of diabetes has received more and more attention [4,5].More and more evidence suggest that many patients with type 2 diabetes can follow a nonalbuminuric pathway to renal function loss, even after accounting for the use of renoprotective agents [6,7].It is important to make clear the relationship between high glucose intake and formation of diabetes and stop the progression of prediabetes into diabetes.
Hyperglycemia is critical in the genesis of diabetic complications. Poor glycemic control is an independent predictor of the development and progression of complications.The tight control of glucose concentration is determined by a balance between glucose absorption from the intestine, glucose production by the liver, and glucose uptake from the plasma.
The liver plays a major role in the regulation of glucose metabolism.The relationship between T2DM and chronic liver diseases (CLD) was discussed actively in recent years.Patients with diabetes have an increased risk of developing CLD.Steatosis, portal brosis, and diabetic steatonecrosis are frequent patterns of hepatic involvement that take place during diabetes [8]. Patients withT2DM are prone to develop nonalcoholic fatty liver disease (NAFLD), and NAFLD itself is associated with a doubled risk of incident T2DM.Many patients with CLD have abnormal glucose metabolism ultimately leads to impaired glucose tolerance and the development of diabetes.The pathogenesis of impaired glucose metabolism during chronic liver disease has not yet been fully understood.The potential of targeting the liver to normalize blood glucose levels has not been fully exploited [9][10][11]. The molecular mechanisms controlling hepatic gluconeogenesis and glycogen storage are not very clear.Further clinical and experimental studies should clarify this issue.We were to make clear how long will it take for high glucose to induce liver lesion.
Intestinal mucosa is the position of oral glucose get into blood.The gut microbiota is a large and complex microbial ecosystem that maintains the homeostasis of the body and environment.The microbiota consumes carbohydrates that are not easily digested by the body.Human gut microbiome is a promising target for managing T2DM.Short-chain fatty acids (SCFAs) are produced by the fermentation of dietary ber by the gut microbiota and are bene cial to the health of the body. Insu cient SCFAs productions are associated with T2DM [12,13].This indicated that Intestinal mucosa and gut microbiota play an important role in the glycemic control.But the change of Intestinal mucosa and gut microbiota after a short time of high glucose intake is not clear.
Sugar consumption is regarded as a major risk for the development of obesity.Diets enriched in sugars including the intake of sugar-sweetened beverages have been consistently linked to the increased risk of obesity, T2DM, and cardiovascular disease. Dietary glucose increased serum glucose and insulin concentrations in the postprandial state.Studies in experimental animals and in man have demonstrated that chronic elevation in the plasma glucose concentration impairs insulin action.Chronic hyperglycemia causes insulin resistance, but the short time glucotoxicity and the underlying mechanisms are unclear [14,15].
High glucose is a critical factor for formation and progression of diabetes.But in which way it induces diabetes, and which organ triggers diabetes are not very clear till now.Most studies are restricted to effects of sugars only on oral high glucose or high glucose infusion on only one or two organs.Therefore, we aim to evaluate the different in uence of short time oral high glucose or high glucose infusion sugars on different organs at the same. We fed or infused liquid high glucose, and analyzed their in uence on liver,pancreas, kidneys,intestine as well as the development gut microbial dysbiosis at same time.

Experimental Animals
This study was performed with 30 male speci c pathogen-free (SPF)-grade Sprague-Dawley rats, aged 12 weeks and weighing approximately 200 ± 20 g.
Rats were purchased from Experiment Animal Center of Guangdong Province. (approval No: SCXK (Yue)-2013-0002, Guangdong, China) . All rats were raised under a light-controlled condition (12 h light/12 h dark) and in a temperature-controlled (23 ± 2 •C) room with food and water available.
All animal experiments were conducted in accordance with the committee guidelines of the Guangdong Provincial People , s Hospital and approved by the Institutional Animal Care and Use Committee of Guangdong Provincial People , s Hospital, No.KY-D-2019-082-01.

Experimental Design
After adaptive feeding, all rats were randomly assigned into three groups, normal diet,ND, n=10; oral high glucose intake,OHG, n=10; high glucose infusion,IHG, n=10. The OHG group rats were fed with 50% high glucose at a dose of 2.5g/kg/day for 2 weeks. The IHG group rats were treated with 50% high glucose by infusion at a dose of 2g/kg/day for 2 weeks.The normal diet group received an equivalent amount of saline for the same period. During the experiment, all rats was received the same standard chow. Fasting blood sugar level (FBS) was measured by a glucometer (Abbot, Alameda, CA, USA) every week in all animals. After 4 weeks of treatment, all rats were sacri ced; 10% chloral hydrate (3 mL/kg i.p., Sigma, St. Louis, MO, USA) was used to reduce pain. The following samples were collected from the rats:Feces were collected individually for at least 3 days for each animal.Blood samples were collected by the retro-orbital plexus for biochemical assays.Kidneys, intestine, pancreas,and liver samples were also collected.

Detection of cytokines and chemokines
In order to evaluate the level of in ammation in rats, ELISA was used to measure serum levels of proin ammatory cytokines interleukin(IL)-6 and tumor necrosis factor (TNF)-a according to the anufacturer's instructions (ELISA, Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Twenty-four-hour microalbuminuria was also assessed by ELISA, according to the manufacturer's instructions.

Histopathological examinations
The small intestine was removed and divided longitudinally, washed and placed in complete media.Sections of intestine, liver, and epididymal white adipose were xed in 10% formalin, para n embedded, and stained with hematoxylin and eosin. Oil Red O staining was performed on frozen sections xed in 4% paraformaldehyde and sucrose protected. For comparison of histologic differences, three sections were blindly selected per sample from each mouse and quanti ed using ImageJ software.

Bioinformatics analysis
Raw data were further ltered according to the following rules using FASTP (https://github.com/OpenGene/fastp):(1) Removing reads containing more than 10% of unknown nucleotides (N);(2) Removing reads containing less than 80% of bases with quality (Q-value) 20. Paired end clean reads were merged as raw tags using FLSAH(version 1.2.11) with a minimum overlap of 10bp and mismatch error rates of 2%. Noisy sequences of raw tags were ltered by QIIME (version 1.9.1 ) pipeline under speci c ltering conditions to obtain the high-quality clean tags. Clean tags were searched against the reference database (http://drive5.com/uchime/uchime_download.html) to perform referencebased chimera checking using UCHIME algorithm (http://www.drive5.com/usearch/manual/uchime_algo.html). All chimeric tags were removed and nally obtained effective tags were used for further analysis. The effective tags were clustered into operational taxonomic units (OTUs) of ≥ 97 % similarity using UPARSE pipeline. The tag sequence with highest abundance was selected as representative sequence within each cluster. Between groups Venn analysis was performed in R project (version 3.4.1) to identify unique and common OTUs. The representative sequences were classi ed into organisms by a naive Bayesian model using RDP classi er(version 2.2) based on SILVA Database (https://www.arb-silva.de/), with the con dence threshold values ranged from 0.8 to 1. The abundance statistics of each taxonomy were visualized using Krona (version 2.6). Biomarker features in each group were screened by Metastats (version 20090414) and LEfSe software [9] (version 1.0).Chao1, Simpson and all other alpha diversity index were calculated in QIIME. OTU rarefaction curve and rank abundance curves were plotted in QIIME. Alpha index comparison between groups was calculated by Welch's t-test and Wilcoxon rank test in R project. Alpha index comparison among groups was computed by Tukey's HSD test and Kruskal-Wallis H test in R project. Weighted and unweighted unifrac distance matrix were generated by QIIME. Multivariate statistical techniques including PCA (principal component analysis), PCoA (principal coordinates analysis) and NMDS (non-metric multi-dimensional scaling) of (Un) weighted unifrac distances were calculated and plotted in R project. Statistic analysis of Welch's t-test, Wilcoxon rank test, Tukey's HSD test, Kruskal-Wallis H test, Adonis (also called Permanova) and Anosim test was calculated using R project.The KEGG pathway analysis of the OTUs was inferred using Tax4Fun(version 1.0).

Short time high glucose led to persistent hyperglycemia
In order to compare the in uence of high glucose infusion and oral high glucose on blood glucose.Fasten blood sugar level (FBS) was measured before glucose taking,14days after glucose taking,7 days and 14 days after ceasing of glucose taking.Both oral high glucose and high glucose infusion increased blood glucose obviously.The blood glucose decreased slowly 7 days after ceasing of glucose taking in both groups.The rats blood glucose in both groups was higher than normal diet rats till 14 days after ceasing of high glucose.There is no difference between oral high glucose intake and high glucose infusion. See gure 1.

Short time high glucose induced in ammation
At the end of high glucose intake,serum levels of in ammatory cytokines IL-6 and TNF-a were measured to evaluate the level of in ammation in rats. Oral high glucose and high glucose infusion increased serum levels of IL-6 and TNF-a obviously.High glucose infusion was a little more intensive than oral high glucose.See table 1.

Short time high glucose increased hepatic lipid accumulation
Hepatic fat accumulation was investigated 14 days after ceasing of high glucose.Both oral high glucose and high glucose infusion induced obvious steatosis in rats liver.A large number of hepatocytes were injured.There is no difference between oral high glucose intake and high glucose infusion.See gure 2.

Short time high glucose induced damage of intestinal mucosa
Small intestinal mucosa of rats were collected for pathological tests.Both oral high glucose and high glucose infusion induced the small intestinal mucosa injury. Oral high glucose led to swelling and necrosis of individual intestinal villi.Some epithelial cells disappeared after 2 weeks of oral high glucose.High glucose infusion led to necrosis and disappearence of cells in the upper layer of intestinal mucosa.The lesion was con ned to the mucosa.See gure 3.

Changes of gut microbiota
To elucidate the mechanism of the effect of high glucose, we investigated the impact of high glucose on the gut microbiota in SD rats. 16S rDNA sequencing was used to assess changes in the fecal microbiota of SD rats with oral high glucose or high glucose infusion.Akkermansiaceae Lactobacillaceae Muribaculaceae Ruminococcaceae Peptostreptococcacea and Clostridiaceae_1 account for 60% of gut microbiota in SD rat.Rats in two groups lost gut microbial diversity.It was characterized by a lower proportion of Akkermansiaceae and a markedly increased proportion of Lactobacillaceae,Muribaculaceae, Ruminococcaceae,Peptostreptococcacea.

Discussion
Sugar consumption is regarded as a major risk for the development of obesity and diabetes.Prediabetes or intermediate hyperglycemia is a high risk state for developing T2DM [16,17].We compared the effect of oral high glucose and high glucose infusion on blood glucose.Both of them increased blood glucose obviously.And they led to persistent hyperglycemia till two weeks later.They both delayed the weight gain of rats.These results con rmed that sugar is a critical cause of diabetes. We need to reduce the intake of sugar.
In ammation involves a tightly regulated process, divided into two complementary subsystems: the innate immune system and the highly adaptive immune system. More and more study indicated that diabetes is a kind in ammation. It is now accepted that the obesity-associated chronic, low-grade systemic in ammation is a major underlying factor for the development of many metabolic diseases [18].Our results indicated that only 2 weeks of oral high glucose or high glucose infusion induced in ammation in rats.
Clinical and pathophysiological studies have shown type 2 diabetes to be a condition mainly caused by excess fat accumulation in the liver and pancreas. Excess fat worsens hepatic responsiveness to insulin, leading to increased glucose production.Removal of excess fat from liver via substantial weight loss can normalise hepatic insulin responsiveness [19,20].Negative energy balance in type 2 diabetes causes a profound fall in liver fat content resulting in normalisation of hepatic insulin sensitivity within 7 days.As the period of negative energy balance extends and liver fat levels fall to low normal, the rate of export of triacylglycerol from the liver falls.The primary care-based Diabetes Remission Clinical Trial showed that 46% of people with type 2 diabetes could achieve remission at 12 months, and 36% at 24 months, mediated by weight loss.Glucotoxicity and lipotoxicity are key features of type 2 diabetes mellitus, but their molecular nature during the early stages of the disease remains to be elucidated [21,22].Our study indicated that both oral high glucose and high glucose infusion induced obvious steatosis in rats liver in just 2 weeks.But there was not obvious lesion on kidneys and pancreas.Liver may be the rst organ damaged by high glucose.Is liver the trigger of diabetes?We need further study.
Intestinal epithelium is characterized by its remarkable self-renewal ability.The crypt base columnar cells marked by Lgr5 represent the actively proliferating stem cells that mediate the daily renewal of intestinal epithelium.The constant renewal cycle takes place in a hostile environment characterised by the presence of bacterial toxins and metabolites,dietary antigens and mutagens, and immunological cytokines and oxidative stress.The intestine has been implicated as a key organ that critically contributes to the development of obesity-associated chronic in ammation and systemic insulin resistance, and metabolic dysregulation [23,24].Glucose directly stimulates intestinal epithelial cells [25].Our results indicated that both oral high glucose and high glucose infusion led to the small intestinal mucosa injury.The damage from oral high glucose was more serious than high glucose infusion.It in uenced the villi and mucosa at the same time.This may be the result of direct contaction.
The gastrointestinal (GI) tract is a highly complex organ composed of the intestinal epithelium layer, intestinal microbiota, and local immune system.Gut microbiota is the most diverse communities.It is in constant interaction with our body's cells and systems.Different sections of the GI tract contain distinct proportions of the intestinal microbiota [26][27][28]. Gut microbiota and its metabolites play pivotal roles in host physiology and pathology.Diet is one of the various factors that in uences the microbiota.Intestinal microbiota modulate metabolism and associate closely with epithelial cells in the intestine [29,30].The intestinal microbiota converts ingested nutrients into metabolites that target either the intestinal microbiota population or host cells.As metabolite of intestinal microbiota,Short Chain Fatty Acids(SCFAs) are a major energy source for intestinal epithelial cells in the colon and reinforce the intestinal barrier function via multiple mechanisms.SCFAs can reduce in ammation and protect kidney.But lipopolysaccharide (LPS) comes from microbiota induces in ammation and injury of kidney,heart,cerebrovascular and other organs.A vast number of studies have demonstrated a remarkable role for the gut microbiota and their metabolites in the pathogenesis of T2DM.Accumulating evidences suggest that SCFAs regulate in ammation, energy metabolism, and blood pressure, which affects kidney function through the gut-kidney axis [31][32][33].Our study indicated that rats treated with high glucose lost gut microbial diversity in two weeks.It was characterized by a lower proportion of Akkermansiaceae and a markedly increased proportion of actobacillaceae, Muribaculaceae,Ruminococcaceae, Peptostreptococcacea.Decreasing of Akkermansiaceae is closely related with fat and diabetes.

Conclusion
We may conclude that high glucose induce lesion of liver and intestinal epithelium, and gut microbial dysbiosis.All these induce in ammation and trigger diabetes.

Declarations
Ethics approval and consent to participate Written informed consents were obtained from all participants and this study was permitted by the Ethics Committee of Guangdong Provincial People , s Hospital. Tables   Table 1 High glucose increased blood IL-6 and TNF-α (pg/ml) , P < 0.01 compared with the control group.
Compared to control group,blood IL-6 and TNF-α of rats increased obviously after 2 weeks of oral high glucose and high glucose infusion. Figure 1 Change of blood glucose Blood glucose of rats in oral high glucose intake(OHG) group and high glucose infusion(HGI) group was obviously higher than control group(NC) till 14 days after ceasing of glucose taking.There is no deference between oral high glucose intake and high glucose infusion Damage of glucose on liver A. liver of control group. X200 B. liver of oral high glucose group. X200 C. liver of high glucose infusion group. X200 Both oral high glucose and high glucose infusion induced obvious steatosis in rats liver.  Gut microbiota change after high glucose intake Relative abundance of gut microbiota in SD rats.

Figures
N,control group. M1,oral high glucose group. T1,high glucose infusion group.