Type 1 Diabetes and Dysfunctional Intestinal Homeostasis

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Review

Type 1 Diabetes and Dysfunctional Intestinal Homeostasis Francesca D’Addio1,2 and Paolo Fiorina1,2,* Despite the relatively high frequency of gastrointestinal (GI) disorders in individuals with type 1 diabetes (T1D), termed diabetic enteropathy (DE), the pathogenic mechanisms of these disorders remain to be elucidated. While previous studies have assumed that DE is a manifestation of diabetic autonomic neuropathy, other contributing factors such as enteric hormones, inflammation, and microbiota were later recognized. More recently, the emerging role of intestinal stem cells (ISCs) in several GI diseases has led to a new understanding of DE. Given the absence of diagnostic methods and the lack of broadly efficacious therapeutic remedies in DE, targeting factors and pathways that control ISC homeostasis and are dysfunctional in DE may represent a new path for the detection and cure of DE. The Rise of Diabetic Enteropathy In 1966 the first report of an individual with diabetes suffering from gastrointestinal (GI) disorders, which included diarrhea, constipation, abdominal pain, and fecal incontinence, appeared in the literature [1]. Interestingly, these abnormalities were not solely associated with neuropathy or with other diabetic complications, while other difficult-to-assess intestinal disturbances (e.g., celiac disease) were virtually excluded. In particular, diarrhea and constipation, the most common symptoms reported in the study, were almost always present, and were accompanied by colonic dilatation and absence of villi in the small intestine, suggesting abnormalities in the mucosa [1]. In a later study of 138 diabetic individuals, 76% of diabetic individuals surveyed for the presence of GI symptoms described suffering from gastroparesis (delayed gastric emptying), diarrhea, constipation, and fecal incontinence [2]; all symptoms correlated with altered GI motility. Moreover, in this same study, the authors demonstrated maladaptive neurohormonal colonic signaling triggered by food ingestion [2], suggesting a potential link between abnormal motility and disruption of the intestinal mucosa and local crypts. Therefore, study of other pathogenic mechanisms such as bacterial overgrowth, malabsorption, and abnormal response to food intake was also recommended [2,3]. In 2001, a study conducted on 8657 diabetic individuals recognized that GI symptoms represent a diabetes complication [4]. In 2002, in a cohort of 1101 subjects with either type 1 (T1D) or type 2 (T2D) diabetes, analysis of GI symptoms assessed a link with the occurrence of other diabetic complications (e.g., nephropathy, retinopathy, neuropathy), and established that poor glycemic control was an independent risk factor for the onset of GI disorders [4,5]. Furthermore, a similar report also documented that diabetic individuals with increased GI symptoms experience reduced quality of life [6]. Finally, in the same cohort of subjects, duration of diabetes and type of diabetes did not correlate with the development of GI symptoms, while Ko et al. reported an association between duration of diabetes with self-reporting GI symptoms in individuals with T2D, which occurred over a period of 8 years (median of 2 years) from diagnosis. This suggests a similar behavior of DE compared with other diabetes-related complications, and strengthens the role of prolonged hyperglycemia

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Trends Diabetes is a multiorgan disease that also targets the gastrointestinal (GI) tract, leading to the development of diabetic enteropathy (DE). DE is poorly characterized thus far, despite its high frequency in both type 1 and type 2 diabetic individuals. Recent evidence suggests that DE as well as other GI diseases may arise due to an alteration in intestinal stem cell (ISC) homeostasis. Because few diagnostic and therapeutic approaches are available in DE, targeting novel mechanisms of disease such as disruption of ISCs is essential. Considering the difficulties encountered by cell therapy, targeting hormonal-like axes that control ISCs and their homeostasis, such as with ectoTMEM219, may represent a novel strategy to treat GI diseases and DE.

1 Nephrology Division, Boston Children's Hospital, Harvard Medical School, Boston, MA 02115, USA 2 Transplant Medicine, IRCCS Ospedale San Raffaele, Milan 20132, Italy

*Correspondence: [email protected] (P. Fiorina).

http://dx.doi.org/10.1016/j.tem.2016.04.005 © 2016 Elsevier Ltd. All rights reserved.

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as risk factor [3]. Although, the number of individuals with T1D enrolled in all the aforementioned studies was very small and type of diabetes was rarely included in the analysis as a separate risk factor, thus the impact of T1D on GI disorders could be hardly recognized. In summary, in the past 20 years, different studies have demonstrated that the significance of GI disorders in T1D and generally in diabetes has often been underestimated in both their frequency and in their level of contribution to the severity of diabetic complications [3,7,8]. GI disorders should in fact be recognized as an independent diabetic complication, termed diabetic enteropathy (DE, see Glossary), and the treatment of DE must be considered in the clinical management of individuals with T1D.

A Largely Unknown Etiopathogenesis The multifactorial pathogenesis of DE has been demonstrated by many reports [2,9,10] and hyperglycemia-associated neuropathy was considered a major player in this pathogenesis [11,12]. Autonomic neuropathy is regarded as a major cause of intestinal motility dysfunction in T1D because neural fibers are damaged by both oxidative stress and inflammation, thus favoring neural degeneration and impaired neural regeneration [13,14]. However, other factors in addition to enteric nervous system may serve to control intestinal motility, including enteral hormones, smooth muscle cells, interstitial cells of Cajal (ICCs), and gut microbiota, which may all be altered in T1D, thereby favoring DE development. Among enteric hormones, incretinrelated peptides (e.g., GLP-1, GLP-2, PYY) coordinate the intestinal response to food intake, but they may also have a protective role on enteric neurons [15,16]. Serotonin and somatostatin, released upon mechanical and chemical stimulation, have been classified mainly as local neurotransmitters that activate GI motility, but they may serve also as growth factors for enteric neurons and fibers [17,18]. Alterations in smooth muscle cells as well as in ICCs have been well documented in animal models of diabetes, but their relevance in human subjects and the mechanisms behind their occurrence need to be clarified [18,19]. In T1D individuals with a long history of T1D and major complications, such as end-stage renal disease (ESRD), intestinal disorders have been primarily linked to the effect of malnutrition, diet restriction, and ongoing therapy, but none of these factors appeared determinant [20]. It has been recently suggested that diabetes may affect GI motility by altering gut microbiota, thus impairing the release of local neurotransmitters and hormones, which in turn compromise intestinal barrier permeability and function [21]. Furthermore, alterations in gut microbiota as well as inflammation associated with diabetes itself may trigger production and release of inflammatory cytokines [e.g., interleukin (IL)1b, interferon (IFN)-g, IL-4, IL-18], which target the intestinal epithelial compartment as well as enteric neurons [21], thereby promoting local inflammation and contributing to the onset of DE. It has also been suggested that the autoimmune response in T1D may involve the intestine and its peptidergic neurons, resulting in the production of cytokines, inflammation, and tissue degeneration [22,23]. More recent studies also hypothesized a role for altered intestinal epithelial renewal in the pathogenesis of DE, as observations of the intestinal mucosal morphology in diabetic rodents revealed a significant local tissue rearrangement [24,25], which was also associated with altered colonic transit [26]. Noteworthy, other colorectal diseases, with similar features to DE, may originate from disruption in intestinal homeostasis due to an alteration in the self-renewing local epithelial compartment, thus pointing to this mechanism also accounting for DE [25,27,28]. In this regard, diabetes, primarily T2D, has been also associated with the risk of developing colorectal cancer (CC) among other colorectal diseases [29,30], thus reinforcing the link between diabetes and intestinal dysfunction. However, while various epidemiological observational studies supported this finding [29,30], the mechanism whereby CC may arise in T2D individuals remains to be established. The effective role of enterotrophic factors (e.g., insulin and insulin-like growth factors) in altering the intestinal epithelial turnover and local stem cells function in the course of diabetes needs to be further explored [31]. Moreover, as stem cell-driven tissue renewal is required to maintain intestinal epithelium due to the persistent epithelial exposure to environmental injury and physiological stress [24,27], alterations in

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Glossary Circulating enterotrophic factors/ hormones: systemic factors such as hormones that may serve to control stem cell homeostasis in the crypts of the small and large intestine, the levels of which may be altered during disease conditions. Diabetic enteropathy (DE): gastrointestinal disorders with high frequency in T1D and T2D, which include diarrhea, constipation, abdominal bloating and pain, and fecal incontinence. Diabetes targets the intestine as well as many other organs and tissues in humans. Ecto-TMEM219: a novel recombinant protein generated and cloned from the extracellular domain of the IGFBP3 receptor (TMEM219), which quenches peripheral IGFBP3 and prevents binding of IGFBP3 and TMEM219 on ISCs, as well as preventing IGFBP3-mediated apoptosis. Gastrointestinal (GI) diseases: GI diseases other than DE, with shared symptoms and/or a probable shared ISC-based origin, but with different triggers with regard to how ISCs are targeted. They include colorectal precancerous lesions and cancer, celiac disease (CD), inflammatory bowel diseases, infectious diseases, irritable bowel syndrome, and intestinal drug toxicity. Gastrointestinal Symptom Rating Scale (GSRS) and Diabetes Bowel Symptom (DBSQ) Questionnaires: questionnaires assessing the occurrence of GI symptoms in the general population and in a targeted disease population, self-administered or administered by medical staff. Their purpose is to allow for evaluation of the prevalence of GI symptoms by using an anonymous and clear format. Insulin-like growth factor binding protein 3 (IGFBP3): a circulating binding protein released by the liver that functions as a carrier for IGF-I and regulates its bioavailability. Alone, IGFBP3 binds its receptor TMEM219, which is expressed on ISCs, and mediates cell apoptosis. Hyperglycemia and inflammation may increase hepatic release of IGFBP3 in diabetes. Intestinal stem cells (ISCs): stem cells residing at the crypt base and expressing LGR5 and EphB2 markers, that differentiate into other cell types along the crypts

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intestinal mucosa seen in individuals with DE may forecast the occurrence of GI symptoms and may arise from a defect in the control of local stem cells. The failure of local stem cells in differentiating along the crypts into other cell types, particularly into enteroendocrine cells (i.e., GLP-1-, GLP-2-, and serotonin-releasing cells) may reduce the local neurohormonal protective effect on enteric nerves exposed to hyperglycemia as well as prevent their regeneration, thereby contributing to the development of enteric neuropathy and DE [16]. To summarize, all the aforementioned factors may interact and account for the development of DE, but the major determinant in the pathogenesis of DE remains to be established.

The Current Landscape in the Diagnosis and Treatment of Diabetic Enteropathy

(enteroendocrine cells, goblet cells, and enterocytes) as well as maintain normal self-renewal properties and intestinal homeostasis. Paracrine factors modulate and control their fate. Mini-guts: 3D structure organoids obtained by culturing intestinal crypts or single stem cells that recapitulate the major features of intestinal homeostasis and represent proxies of crypts and stem cell activity and function.

While recent publications [4,7,32–35] support the clinical relevance of diabetic enteropathy as a novel diabetic-related disease and its role in increasing the morbidity of individuals with diabetes (Table 1), reliable diagnostic tools and therapeutic options remain elusive. The Diagnostic Uncertainty of DE The Gastrointestinal Symptom Rating Scale (GSRS) Questionnaire has been extensively used in the past and is currently used by gastroenterologists to assess the prevalence of GI symptoms in the general population [36]. In 2003, the Diabetes Bowel Symptom Questionnaire was validated and specifically addressed GI symptoms related to diabetes [37], although as a self-administered screening instrument, it may have several limitations (e.g., rate of response,

Table 1. Evidence from Clinical Retrospective Studies Linking Diabetes with GI Symptomsa[2_TD$IF] Study Design

Subjects (n)

Diabetes

Main Findings

Refs

Upper/lower GI symptoms: questionnaire. GI radiological exams.

138

T1D, T2D

76% with GI symptoms. Constipation: 60%. Relationship with diabetic autonomic neuropathy.

[2]

Upper/lower GI symptoms: questionnaire. Diabetes status. Results were compared with CTRL.

333

T1D, T2D

T2D is associated with GI symptoms.

[83]

Upper/lower GI symptoms: interviews. Diabetes status (HbA1C, glycemia). Occurrence of other complications.

149

T2D

70% with GI symptoms. Duration of diabetes is associated with GI symptoms.

[3]

Lower GI symptoms: questionnaire. Diabetes status (HbA1C, glycemia). Results were compared with CTRL.

8567

T1D, T2D

GI symptoms negatively impact health-related quality of life in diabetes.

[4]

Upper/lower GI symptoms: DBSQ. Complications of diabetes (clinical history) and self-reported glycemic control. HbA1C measurement.

1101

T1D, T2D

GI symptoms in diabetes may be linked to diabetic complications and to poor glycemic control.

[5]

Quality of Life: Short-Form 36. Lower GI symptoms: questionnaire. Self-reporting diabetes status.

1101

T1D, T2D

GI symptoms negatively impact health-related quality of life in diabetes.

[6]

Lower GI symptoms: GSRS, anorectal manometry, histology (sigmoidoscopy). Diabetes status (HbA1C, glycemia). Comparison with CTRL.

60

T1D

High frequency of GI symptoms in T1D. Abnormalities in anorectal sphincter, mucosa, ISCs. High IGFBP3 serum level.

[35]

[3_TD$IF]a

Abbreviations: GI, gastrointestinal; GSRS, Gastrointestinal Symptom Rating Scale Questionnaire; T1D, type 1 diabetes; T2D, type 2 diabetes; HbA1C, glycated hemoglobin; DBSQ, Diabetes Bowel Symptom Questionnaire; CTRL, healthy subjects; ISCs, intestinal stem cells; IGFBP3, insulin-like growth factor binding protein 3.

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self-evaluation, compliance, etc.). Anorectal manometry has been suggested as a technique to further confirm altered motility of the lower tract of the intestine [38], and colonic transit studies can be performed to assess motility dysfunction [39]. Moreover, GI endoscopy can be used to exclude other known GI diseases related to similar symptoms (e.g., inflammatory bowel disease, colonic polyposis, and celiac disease) [40]. Use of new endoscopy techniques such as wireless video capsule endoscopy (WCE), device-assisted enteroscopy (DAE), chromoendoscopy, and confocal endomicroscopy has significantly improved the diagnosis of GI disorders, particularly that of inflammatory bowel disease (IBD), and has also facilitated monitoring of therapy and relapse [41]. Despite the progression achieved, the need for noninvasive biomarkers in the diagnosis of GI diseases led to development of new strategies and focused on fecal biomarkers. Fecal immunochemical testing (FIT) and stool DNA (sDNA) are being currently tested to support the diagnosis of colorectal cancer [42]. Fecal detection of calprotectin, lactoferrin, and S100A12, markers of inflammation, has been reported in the diagnosis of IBD [43], while detection of calprotectin alone represents a specific fecal marker for irritable bowel syndrome (IBS) [44]. As a noninvasive approach, brain functional magnetic resonance imaging (MRI) and magnetic resonance enterography have been tested in the diagnosis of IBS and in IBD, respectively, to distinguish individuals with brain-related functional or intestinal morphological GI disorders from healthy subjects, with encouraging preliminary results [45,46]. Unfortunately, there are neither serological, fecal, or urinary screening markers currently available for the diagnosis of DE nor specific morphological lesions, and therefore the most important step in the diagnostic process of identifying an individual who may develop DE remains the evaluation of medical history and exclusion of other common causes of GI symptoms. The Therapeutic Vacuum of DE Considering that poor glycemic control is a risk factor for all diabetic complications, reducing glycated hemoglobin levels (HbA1C) to within the ranges suggested by the American Diabetes Association guidelines represents a first line of intervention that facilitates the prevention of all major diabetic complications, including DE [4,47]. However, the administration of some medications that controls diabetes such as metformin, particularly in T2D, may in fact favor the development of DE [48]. Despite this, the use of drugs for symptom relief represents the most common strategy adopted in DE [23]. Unfortunately, GI symptoms may occur simultaneously (e. g., diarrhea and constipation), and thus the use of GI medications (e.g., loperamide, diphenoxylate, and other antidiarrheal drugs) is indicated to avoid more serious GI complications or hospitalization [8]. Probiotic administration is useful considering potential alterations in the microbiota, while the use of antibiotics remains the treatment of choice for bacterial overgrowth-related diarrhea [49]. Encouraging results in the long-term have been observed in diabetic diarrhea using octreotide (a somatostatin analog), although several side effects have been documented [50]. Finally, in individuals with T1D, pancreas and islet transplantation, capable of restoring near-normal glycemic control with significant benefit in the outcome of major diabetic complications [51–54] may also be considered an advantageous treatment for DE. The successful use of conventional [e.g., corticosteroids, aminosalicylates, antitumor necrosis factor / (anti-TNF/)] and novel anti-inflammatory therapies such as anticytokine drugs (e.g., Infliximab, Adalimumab, and Ustekinumab) and anti-cell adhesion molecules (e.g., Vedolizumab, Natalizumab) in IBD may suggest a potential benefit also in DE, although inflammation is not always observed in the intestinal mucosa of subjects with DE [55,56]. Likewise, given the autoimmune nature of T1D, immunosuppression (azathioprine, methotrexate) may prevent the infiltration and migration of activated peripheral immune cells (T cells, monocytes, eosinophils) to the intestine, as in other autoimmune GI diseases, thus blocking an exacerbating factor in the progression of DE [55]. However, both anti-inflammatory and immunosuppressive drugs have several adverse side effects, which need to be taken into account when administering these agents to individuals with DE, given the marginal role of inflammation and immune activation in its pathogenesis.

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The Emerging Role of Intestinal Stem Cells in DE The multifactorial pathogenesis of DE thus far described, together with the lack of a recognized major factor to be targeted in diagnostic and therapeutic procedures, led investigators to explore whether systemic factors or hormones may be involved in the development of DE (Figure 1, Key Figure). Furthermore, GI symptoms reported in individuals with DE are similar to those reported in other colorectal diseases, therefore the role of intestinal stem cells (ISCs) in various colorectal diseases has been investigated in a number of studies (Table 2). ISCs, located

Key Figure

Key Players Involved in Diabetic Enteropathy Key: Systemic hormones

Type 1 diabetes and intesnal disorders

Enteral hormones Cytokines

GLU

↓ Glycemic control ↑ Oxidave stress ↑ Immune acvaon ↑ Inflammaon

Glucose Bacteria Immune cells Neural fibers

Lymphac system

Blood vessel

Smooth muscle cells

↑ ↓ Enteral hormones

Liver Pancreas Intesne

↓ ICC ↓ Neural regeneraon Microbiota dysregulaon

↓SMCs

GLU GLU GLU

Intesnal crypts ↓ ↓ ISCs ↓ EECs ↓ Epithelial proliferaon

Figure 1. Diabetic enteropathy (DE) arises from a complex interplay between systemic and local factors altered in the course of diabetes. Poor glycemic control favors development of oxidative stress and triggers inflammation, which in turn promotes immune activation. Immune activation may also result from autoimmune responses in type 1 diabetes (T1D). Immune cells, inflammatory cytokines, and reactive oxygen species (ROS) may target peripheral enteric neural fibers and neurons, particularly the interstitial cells of Cajal (ICCs), thus preventing local neural regeneration. Also, smooth muscle cells (SMCs) located in the muscularis mucosae layer may be affected by hyperglycemia, inflammation, immune activation, and oxidative stress. Furthermore, in the course of diabetes, alterations in the gut microbiota may also trigger inflammation of the intestinal mucosa and reduce nutrient absorption, which perpetuates hyperglycemia, impairs enteric hormone release in response to food uptake, and increases permeability of the intestinal barrier. Recent evidence that intestinal stem cells (ISCs) control intestinal homeostasis by proliferating along the crypts and differentiating into enterocytes, goblet cells, and enteroendocrine cells (EECs) shed light on a novel mechanism whereby the intestinal barrier may be disrupted. In particular, systemic and paracrine factors altered in the course of diabetes mediate ISC injury, which causes a reduction in the EEC population, in hormones released by EECs, and in epithelial proliferation, thus leading to loss of intestinal homeostasis and self-renewal properties. This in turn reduces nutrient absorption and response to food intake, thus establishing an ISC– hyperglycemia–hormonal loop underlying the development of DE.

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Table 2. Links between ISCs and GI Disordersa[4_TD$IF] Type of GI Disorder

Role of ISCs

Main Findings

Refs

Inflammatory bowel diseases

Defects in ISC differentiation

Impaired generation of Paneth and goblet cells leads to a defective antimicrobial mucosal barrier

[57,84]

Colorectal cancer

Deregulation and uncontrolled proliferation of ISCs

ISC gene signature and signaling are altered in CC; ISC deregulation predicts CC relapse

[28,60]

Celiac disease

Depletion of ISCs

ISCs favor mucosal healing and clinical remission; ISC altered signaling and deficiency favors CD

[61,85]

Helicobacter pylori gastritis

Manipulation of ISCs Colonization of ISCs

HP colonize and alter turnover of gastric ISCs, favoring glandular hyperplasia

[75]

Precancerous conditions (adenoma, chronic ulcerative colitis, polyposis)

Overpopulation of ISCs

In precancerous conditions, ISC differentiation and turnover are abrogated, resulting in increased numbers of ISCs

[86]

Diabetic enteropathy

Depletion of ISCs Disruption of ISCs

IGFBP3 mediates ISC apoptosis in DE

[35]

[3_TD$IF]a

Abbreviations: GI, gastrointestinal; ISCs, intestinal stem cells; CC, colorectal cancer; CD, celiac disease; HP, Helicobacter pylori; DE, diabetic enteropathy; IGFBP3, insulin-like growth factor binding protein 3.

at the crypt base of the small and large intestine and expressing the signature markers ephrin type B receptor 2 (EphB2) and leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) [28], differentiate into enterocytes, goblet cells, and enteroendocrine cells [57], thus preserving crypt turnover and protecting intestinal homeostasis from environmental injuries. Alterations in ISC regulation and in crypt and epithelial self-renewal properties have been described in IBD [58], in colon precancerous conditions [59], and in colorectal cancer [60]. Recently, it has also been suggested that ISCs may be depleted in active celiac disease, thus leading to impaired regeneration of the intestinal epithelial compartment, which may account for the disappearance of villi [61]. However, other studies conducted in murine models of T2D [62] and obesity [63] documented an increase in ISC self-renewal associated with a shortening of villi in the small intestine, thus suggesting that a disruption of the ISC homeostasis, rather than ISC depletion, may alter other non-ISC populations (e.g., enterocytes) and indirectly affect the villi length in these disease conditions. Further studies are needed to address the link between ISC alteration/depletion and villi morphology. In summary, dysregulation of ISCs and of their niche is associated with loss of intestinal homeostasis (Table 2), thus rendering the intestine more vulnerable to mechanical and chemical stressors. Several agents capable of targeting ISCs, as well as their signaling and their differentiation, have been described in each of the aforementioned conditions (e.g., inflammation in celiac diseases, immune activation in IBD, gene mutations in colorectal cancers) [58,60,61], therefore highlighting ISC-based therapy and tissue engineering as promising treatments to restore intestinal homeostasis in different diseases. Interestingly, transplantation of ISCs or of ISC-derived organoids to regenerate local mucosa has been successfully explored in different preclinical models of colitis [64–66], while attempts at targeting factors and pathways that control ISC turnover are still underway. Nevertheless, there is a growing body of evidence supporting the use of induced pluripotent stem cells and human embryonic stem cells to generate intestinal organoids in vitro that can be engrafted in vivo to reconstitute human intestinal tissue [67], thus paving the way for a novel stem cell-based approach to treat several GI diseases. With regard to DE, it has been suggested that a depletion

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Liver Intesnal Crypt Enterocytes

Enteroendocrine cells Goblet cells

Intesne

Hyperglycemia inflammaon

Stem cells

GLU GLU GLU

↓ ↓ Nutrients absorpon Key: Cytokines GLU

Glucose

↓ ↓ Response to food intake

Mucosa atrophy

IGF-IR

TMEM219

Survival

Altered crypts turnover

Apoptosis Intesnal stem cell

IGFBP3 IGF-I

Figure 2. Diabetes Mediates ISC Dysfunction through an IGFBP3-Mediated Mechanism, Leading to Disruption of Intestinal Homeostasis. Chronic hyperglycemia and inflammation in diabetes alters hepatic release of circulating factors, including reduced production of insulin-like growth factor 1 (IGF-I) and increased release of insulinlike growth factor binding protein 3 (IGFBP3). Excessive release of IGFBP3 into the circulation targets crypts in the small and large intestine by mediating apoptosis of intestinal stem cells (ISCs). The few remaining ISCs therefore fail to differentiate into other cell types along the crypt (e.g., enterocytes, enteroendocrine, and goblet cells), leading to altered intestinal crypt turnover and mucosal atrophy, which ultimately abrogates absorption of nutrients and response to food intake. The resulting malabsorptive state exacerbates both hyperglycemia and inflammation, which in turn maintains hepatic release of IGFBP3, thus establishing an IGFBP3-mediated feedback loop that impairs intestinal homeostasis in diabetes. Abbreviations: IGF-IR, insulin-like growth factor 1 receptor; T1D, type 1 diabetes.

of ISCs in the large intestine was responsible for the GI symptoms and altered mucosa morphology in long-standing diabetic individuals (Figure 2). It was further demonstrated that in the case of DE, a direct apoptotic effect was responsible for ISC loss, particularly in the colon [35]. Reduced expression of major ISC signature markers confirmed that ISCs in the colon mucosa of individuals with DE were almost absent, leading to a failure of regeneration of epithelial crypts and to the impairment of other ISC-derived cells and structures (enteroendocrine cells, neural structures).

New Diagnostic Tools in DE and in Other GI Disorders This recent data confirmed that DE exists at a high frequency in individuals with diabetes of long duration and that it is associated with symptoms that reflect significant morphological changes in the intestine [35]. Flattening of the intestinal mucosa, particularly at the colorectal level, reduction in the number of local crypts associated with an increase in crypt depth and width, and depletion of ISCs were further confirmed as morphological symptoms in a murine model of DE [35], which may represent a signature of DE that can be used for diagnostic purposes when a colorectal bioptic sample is available (Table 3). In line with this, use of the mini-guts model, a 3D culturing system that grows epithelial organoids from single ISCs and recapitulates in vitro the main features of intestinal homeostasis in vivo, may be used as a proxy of ISC activity, thus representing a novel method to test a GI disease condition [28,68,69]. To date, mini-guts have been employed to study cystic fibrosis [70], hereditary intestinal diseases [71], mechanisms

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Table 3. Morphological Features of DEa[5_TD$IF] Morphological Changes

Human Subjects

Macroscopic alterations (increased length and wall cross-sectional area, reduction of physiological curvatures)

Animal Models

Refs

STZ-treated RIP-I/hIFNb transgenic mice STZ-treated rats

[24,87]

Flattening of the mucosa, reduced thickness of the epithelial layer, increased stiffness of the intestinal wall

T1D, T2D

STZ-treated B6 mice with longstanding diabetes NOD mice STZ-treated rats

[1,35,87–89]

Alterations of number, depth, and width of crypts in small and large intestine

T1D

STZ-treated B6 mice with longstanding diabetes NOD mice STZ-treated RIP-I/hIFNb transgenic mice BBdp rats STZ-treated rats

[24,35,87–89]

Shortening, absence, and ultrastructural changes of villi in the small intestine

T1D, T2D

HFD-treated mice (T2D) BBdp rats

[1,62,89,90]

Reduced epithelial proliferation in the large intestine

T1D

STZ-treated B6 mice with longstanding diabetes BBdp rats

[35,89]

Defect of ISCs in number and morphology in the large intestine

T1D

STZ-treated B6 mice with longstanding diabetes

[35]

HFD-treated mice (T2D)

[62]

Increase of ISC self-renewal in the small intestine Reduction of EECs

T1D

STZ-treated B6 mice with longstanding diabetes STZ-treated RIP-I/hIFNb transgenic mice

[24,35]

Signs of neural degeneration in the small and large intestine

T1D

STZ-treated B6 mice with longstanding diabetes STZ-treated RIP-I/hIFNb transgenic mice

[24,35]

[6_TD$IF]a

Abbreviations: DE, diabetic enteropathy; ISCs, intestinal stem cells; EECs, enteroendocrine cells; HFD, high-fat diet; T1D, type 1 diabetes; T2D, type 2 diabetes; STZ, streptozotocin; BBdp, diabetes-prone BB rats; NOD, non-obese diabetic mice.

related to IBD development [72,73], gastric and colorectal malignancies [74], GI infectious diseases [75,76], and DE [35], but their use may be extended, as has been suggested [68,77], to numerous other GI disorders (e.g., irritable bowel syndrome, food intolerance, and malabsorption syndromes, bacterial overgrowth and common intestinal infectious diseases). Moreover, assessment of ISC signature markers, use of mini-guts, and pathological analysis may be used as novel techniques to monitor the effect of medications/drugs that have been administered in the treatment of GI disorders or in the case of suspected drug toxicity that targets the intestine (e. g., immunosuppression therapy, antibiotics). Finally, the fact that circulating enterotrophic hormones, such as insulin-like growth factor binding protein 3 (IGFBP3) [35], may control ISC homeostasis and have been shown to be altered in DE [32], suggests that a screening test measuring levels of these hormones in peripheral serum may help in identifying individuals at risk for developing DE or GI disorders. Additional and further monitoring may also allow for assessment of the efficacy of an administered treatment.

Novel Therapeutic Options for DE Considering that ISC impairment triggers alterations in mucosa morphology and GI motility observed in DE, targeting the mechanism whereby ISCs are disrupted and/or replacing

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damaged/deficient ISCs, may provide a novel approach to counteract the onset and progression of DE. A previous study has demonstrated that treatment of diabetes with pancreas transplantation replenishes impaired ISCs and near-normalizes intestinal mucosa morphology, GI symptoms, and motility dysfunction [35]. Despite the substantial progress made, organ shortages render this and other similar strategies (e.g., islet transplantation) difficult to pursue [78,79]. Moreover, individuals with T2D also suffer from DE [4,5], and in their case, pancreas/islet transplantation is rarely suggested [80]. It has been shown that transplantation of long-term expanded colonic ISCs positive for LGR5 obtained in mice where a colon injury has been induced reestablished the epithelial layer and formed histologically normal self-renewed crypts [66]. Previously cultured and frozen LGR5+ organoids, when transplanted in mice, showed a long-term and well-tolerated engraftment [66], thus suggesting that in vitro expansion and transplantation of ISCs may represent a promising approach for individuals with DE and other GI disorders. Unfortunately, stem cell-based therapy is often marred by difficulties, including generation of cells to be infused, related adverse events, and potential need for immunosuppressive therapy to avoid allograft rejection [81]. Therefore, targeting the mechanisms whereby ISCs are disrupted during DE may offer a more rapid and less invasive approach to be translated to clinical settings. It has been demonstrated that circulating levels of IGFBP3 are increased in individuals with DE and that IGFBP3 directly targets ISCs, thus mediating their loss [35]. In particular, IGFBP3 can prevent insulin-like growth factor (IGF-I) signaling through its receptor (IGF-IR), which controls ISCs, by binding circulating IGF-I and reducing its bioavailability [35,82]. Most importantly, IGFBP3, when massively released in the circulation, acts as an ISC toxic factor, through a proapoptotic IGF-I-independent caspase-mediated mechanism on ISCs, which express TMEM219 (the IGFBP3 receptor) [35]. Furthermore, quenching circulating IGFBP3 with a newly generated molecule based on the extracellular domain of the IGFBP3 receptor TMEM219, ecto-TMEM219, restores self-renewal properties and histological normality of in vitro cultured mini-guts and replenishes ISCs in vivo in murine models of DE, leading to a near-normalization of GI abnormalities [35]. This suggests that ecto-TMEM219 or alternative strategies that block the IGFBP3-mediated detrimental effects on ISCs may be further explored as therapeutic options for DE [32]. Intriguingly, if high IGFBP3 levels are confirmed in other GI diseases, this application may also be extended to other pathological conditions. Finally, other circulating hormones with altered levels in individuals with DE or with GI diseases may be identified in the future. Once these effects on ISCs have been established, targeting those hormones may represent an additional strategy to be explored to expand the horizon for the cure of DE and GI diseases, thus offering our patients the best choice of treatment.

Outstanding Questions What is the clinical relevance of DE in the context of the increasing prevalence of diabetes worldwide? Does DE affect only quality of life or does it also have an impact on the development of other diabetic complications and therefore exacerbate diabetes mortality? Will ISC studies provide new insights in understanding the pathogenesis of DE and allow for design of new diagnostic and therapeutic tools? Do IGFBP3 and more general hormonal axis targeting strategies offer increased clinical benefits in the treatment of DE?

Concluding Remarks and Future Perspectives The growing prevalence of diabetes worldwide will result in increasing proportions of mortality, as well as increased prevalence and associated consequences of other complications of T1D and diabetes in general. DE is a recognized but poorly characterized diabetic complication, and its clinical relevance is increasing due to extensive studies conducted in the diabetic community. While current diagnostic methods and therapeutic options are mainly based on the knowledge of other GI diseases and primarily control GI symptoms, novel approaches exploiting regenerative medicine and stem cell studies are emerging. The multifactorial pathogenesis of DE described thus far is becoming less convincing and demands a new line of investigation that focuses on the role of ISCs and the peripheral hormonal factors that control these cells. In line with this, a novel circulating enterotrophic factor, IGFBP3, also known as enterostaminine, was recently shown to control ISCs in the context of intestinal disorders, thus shedding light on a novel mechanism behind the pathogenesis of DE (Figure 2). Moreover, ISCs have recently become a topic of intense investigation because of their potential as targets for new therapeutic strategies in cancer and intestinal diseases (see Outstanding Questions). In conclusion, the manipulation of enterostaminine using ecto-TMEM219, as we described, will allow for circumvention of costly ex vivo cell expansion and of known problems with cellular sources and culture conditions. This may

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pave the way for novel targeted diagnostic and therapeutic strategies that can be translated from diabetes to other colorectal diseases. Acknowledgments F.D’A. is a recipient of an Italian Scientists and Scholars of North America Foundation (ISSNAF)-Fondazione Marche Fellowship and of a Research Grant from SID-Lombardia 2016 (Finanziamento Ricerca Diabetologica SID Lombardia 2016). P.F. is a recipient of a Minister of Health of Italy grant RF-2010-2314794 and RF-2010-233119. P.F. is the recipient of an EFSD/Sanofi European Research Programme and is supported by an American Heart Association (AHA) Grant-in-Aid.

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