Melvin R. Hayden, MD, Department of Internal Medicine, University of Missouri-Columbia School of Medicine, D109 Diabetes Center, UHC, One Hospital Drive, Columbia, MO 65121-0001 E-mail: firstname.lastname@example.org
Ultrastructural observations reveal a continuous interstitial matrix connection between the endocrine and exocrine pancreas, which is lost due to fibrosis in rodent models and humans with type 2 diabetes mellitus (T2DM). Widening of the islet-exocrine interface appears to result in loss of desmosomes and adherens junctions between islet and acinar cells and is associated with hypercellularity consisting of pericytes and inflammatory cells in T2DM pancreatic tissue. Organized fibrillar collagen was closely associated with pericytes, which are known to differentiate into myofibroblasts—pancreatic stellate cells. Of importance, some pericyte cellular processes traverse both the connecting islet-exocrine interface and the endoacinar interstitium of the exocrine pancreas. Loss of cellular paracrine communication and extracellular matrix remodeling fibrosis in young animal models and humans may result in a dysfunctional insulino-acinar-ductal–incretin gut hormone axis, resulting in pancreatic insufficiency and glucagon-like peptide deficiency, which are known to exist in prediabetes and overt T2DM in humans.
The endocrine and exocrine pancreas has traditionally been considered to be 2 separate entities. However, the pancreas is usually classified as a lobulated compound endocrine tubuloacinar gland. The endocrine (islets) secrete hormones directly into the blood (internal secretion), and the exocrine pancreas secretes enzymes and other substances into a duct system and thus to a body surface (eg, the gut) (external secretion).
Embryologically, both pancreatic entities develop in a similar fashion by invaginations of epithelial cells into the connective tissue underlying an epithelial membrane. The site of original invaginations of the exocrine portion persists as a duct and acinar system, whereas connections with the epithelial membrane are lost in the islet. Thus, islet tissue secretory products are passed into the systemic circulation as hormones.1
The structure of the pancreas in mammals has undergone significant ontologic evolution. For example, in protochordates the islet cells are distributed throughout the gut mucosa, in the Atlantic hagfish the first islet-like structure is noted and closely related to the bile duct, in cartilaginous fish (sharks and rays) the islet becomes intimately associated with the pancreatic duct, and in mammals the islets and exocrine tissue are fused into one compound glandular organ functioning synergistically to aid in the digestion of oral nutrients and metabolism. Thus, evolution has led to the merging of the endocrine and exocrine pancreas into one organ, allowing for improved endocrine and paracrine communication.1
Indeed, an artificial subdivision of the pancreas precludes the study of this lobulated compound tubuloalveolar-acinar gland and precludes the investigation of the endocrine-exocrine pancreas in its role as an interdependent synergistic system in health and disease.2 Therefore, we have chosen to focus on the cell-cell, cell-matrix, extracellular matrix (ECM), insulo-acinar-ductal-portal vascular pathway and the insulo-acinar-ductal-pancreatic enzyme–incretin gut hormone axis communications.
Recently, we have made observations utilizing microscopy (transmission electron microscopy [TEM] and special staining with light microscopy) demonstrating that many of these cell-cell, cell-matrix, vascular, and ductal communications are lost or impaired in young rodent animal models of hypertension, insulin resistance (IR), oxidative stress, and type 2 diabetes mellitus (T2DM) due to fibrosis.3–5
With progressive fibrosis, the normal wound-healing process goes awry because of an ongoing wounding process with resulting structural alterations, which hinders proper tissue and organ function and leads to tissue dysfunction and eventual organ failure. In humans with the cardiometabolic syndrome and T2DM, we have observed that these same communications are lost due to fibrosis of the peri-islet–islet exocrine interface (IEI), the endoacinar and the interlobular periacinar interstitium leading to pancreatic failure.
Endocrine/Exocrine Cell-Cell Communications Are Lost
The IEI is an important anatomic and functional region allowing for cell-cell communication between the endocrine islet and exocrine acinar cells of the pancreas. Recently, we have observed that desmosomes and adherens junctions exist between islet and acinar cells within the IEI. The functional communication between these 2 cell types has not been explored at this time but are structurally lost during the remodeling changes associated with the development of T2DM. The IEI was observed to widen at least 40-fold when comparing the 7-week-old male Leprdb/db (db/db), a leptin receptor–deficient mouse model of T2DM, to its aged-matched, wild-type, lean C57BL/6 nondiabetic littermate controls (db/dbWT) (Figure 1). Insulin and glucose levels were not available for this ultrastructural study; however, it is known that the db/db model develops hyperinsulinemia within 2 weeks, followed by obesity at 3 to 4 weeks and hyperglycemia at 4 to 8 weeks of age.6 Similar remodeling changes have been noted in the Ren2 model (a rat model of hypertension, IR, and oxidative stress harboring the mouse renin gene)3,4 and the human islet amyloid polypeptide (HIP) rat model (a Sprague Dawley rat transfected with the human amylin gene) of T2DM when compared with the age-matched male Sprague Dawley controls.5 The loss of these cell-cell connections (desmosomes and adherens junctions) between the islet and acinar cells have not been previously described, to our knowledge.
We hypothesized that increased oxidative stress may activate IEI matrix–degrading proteases such as matrix metalloproteinases (MMPs) and result in enzymatic degradation of these cell-cell connections; thus, communication between these 2 cell types is lost. For example, matrix-degrading proteases (MMP-2, -12, and -14) are up-regulated in the islets of the Zucker diabetic fatty (ZDF) rat model and contribute to the development of diabetes in these models. Further, MMP inhibitors are capable of abrogating the increased remodeling fibrosis of the islet and substantially prevented diabetes in these models.7
Cell-Matrix Communications Are Lost
Pericapillary remodeling fibrosis and islet amyloid deposition have been identified in the ZDF,7 HIP,5,8,9 and db/db10 models of T2DM. Deposition of collagen and/or islet amyloid in the pericapillary region may result in a diffusion barrier and lost cell-cell and cell-matrix communications to small molecules and islet secretory granules, referred to as endothelial β-cell uncoupling (Figure 2).11
Of importance, the cell-matrix connections and communications were observed to be lost due to widening and subsequent matrix fibrosis in the IEI of the db/db diabetic model (Figure 1).
Matrix Communications Are Lost
The peri-islet interstitium–IEI is a potential space that widens in db/db models, resulting in the loss of cell-cell communication between islet and acinar cells in the pancreas (Figure 1). In both the db/dbWT and the db/db diabetic models, the IEI interstitium is continuous with the endoacinar and interlobular periacinar interstitium of the exocrine pancreas, confirming an insulo-acinar-ductal connection, which may be lost due to remodeling fibrosis (Figure 3).
Hypercellularity of the IEI and Early Fibrotic Changes
The IEI in the db/dbWT model contains a very loose areolar matrix with sparse unorganized collagen and reticular fibers and 4 or 5 capillaries (endothelial cells and their supporting pericytes) providing capillary connectivity and occasional unmylelinated neural cells. In contrast, the IEI in the db/db diabetic model becomes hypercellular, consisting of 12 to 15 pericytes with their long cytoplasmic processes being the primary cell type and a few monocytic inflammatory cells (Figure 4). Early organized fibrillar-banded collagen typical of early fibrosis was noted and closely associated with pericytes, which are known to be capable of differentiating into myofibroblast-like pancreatic stellate cells in other models3,9 (Figure 5).
Pancreatic IEI and Interlobular Periacinar Interstitial Fibrosis
The IEI early fibrosis at 7 weeks in the db/db diabetic model portends the development of significant fibrosis in the IEI and interlobular and endoacinar interstitium in the db/db model as it ages, as well as in other T2DM rodent models and humans with T2DM (Figure 6). For example, by 14 weeks the IEI fibrosis in the db/db diabetic model known to overexpress angiotensin II (Ang II) was markedly increased compared with that in the db/dbWT controls and, interestingly, was abrogated with the angiotensin receptor blocker candesartan.12 In addition, the Ren2 rat model transfected with the mouse renin gene at 9 weeks developed interlobular periacinar interstitial fibrosis, while there was only early organized collagen deposition in the IEI, indicating a temporal-spatial deposition of collagen and fibrosis in this transgenic model of local tissue Ang II overexpression.3,4 The fibrosis in the Ren2 model seemed to originate in the perivascular-periductal areas of the interlobular periacinar interstitium and emanate to the surrounding loose areolar matrix of the exocrine interstitium. The involvement of a local pancreatic renin-angiotensin-aldosterone system is not to be underestimated with regard to the important role it plays in oxidative stress, vasoconstriction, interlobular periacinar, IEI, and eventual endoacinar interstitial remodeling fibrosis.3,4,8,10–18
Irrespective of the etiologic agent(s), pancreatic ECM remodeling fibrosis represents a final common pathway, as in most chronic diseases, leading to destruction of tissue architecture, function, and organ failure as in T2DM.
In the db/db obese diabetic model and in humans with T2DM, there are both islet and exocrine wounding. The major offending agents responsible for this wounding in the db/db diabetic model and other obese T2DM animal rodent models are Ang II; nicotine adenine dinucleotide phosphate (reduced) oxidase activation via the angiotensin type 1 receptor, including up-regulation of the mineralocorticoid receptor; hyperglycemia; and reactive oxygen species (ROS), oxidative stress responsible for activating MMPs and instigating remodeling fibrosis. This chronic wounding results in the persistent activation of the innate classic tissue wound healing response characterized by inflammation, granulation tissue, and matrix formation, including the recapitulation of embryonic genetic memory.3,4,9,13–15
The ensuing matrix fibrosis is associated with loss of ECM elasticity and stiffening, which affects conduit structures including capillaries and exocrine ducts, impaired diffusion of molecules such as nitric oxide and hormones such as insulin, impaired cellular migration such as newly synthesized periductal β-cells, and contraction and parenchymal acinar loss due to replacement fibrous and adipose tissue replacement as depicted in Figure 6. These multiple changes due to the deposition of organized fibrillar-banded collagen result in lost ECM transportation and communications of the intra-islet, peri-islet–IEI, endoacinar, and interlobular periacinar interstitium.
Postmortem histologic changes in the pancreata of human and rodent models (db/db, ZDF, Ren2, and HIP) of T2DM typically include intra-islet, IEI, endoacinar, and interlobular periacinar ECM fibrosis. Concurrently, there exists islet amyloid deposition in the human and HIP models, exocrine and endocrine adipose replacement, loss of β-cell mass due to apoptosis, replacement fibrosis, and islet amyloid deposition. Contemporaneously, there is loss of the exocrine parenchyma due to apoptosis, replacement fibrosis, adipogenesis, and a full spectrum of vascular remodeling changes including arteriosclerosis, arteriolosclerosis, and atherosclerosis (Figure 6).13,16–18 Of importance, when the above findings were carefully scrutinized by statistical analysis the most significant finding was pancreatic interstitial fibrosis with a P value of <.001.18
Pancreatic Pericytes as Pancreatic Stellate Cells
The discussion of fibrosis in the pancreas would be incomplete without discussing the role of the fibrogenic pericyte cell (Figure 4 and Figure 5) and its relation to the profibrogenic pancreatic stellate cell. The pancreatic stellate cell was originally described a decade ago19,20 and has been of topical interest regarding pancreatic fibrosis in humans and various animal models of acute and chronic pancreatitis.21,22 These triangular cells are typically located at the base of acinar cells within the endoacinar and interlobular periacinar interstitium, in contrast to their more elongated appearance within the IEI. The pericytes-pancreatic stellate cells are closely associated with endothelial cells of the microcirculation, where they appear to connect capillaries within the interstitium. Once activated by injurious stimuli such as oxidative stress (ROS) they differentiate into profibrotic cells capable of synthesizing collagen type 1 and III and fibronectin (Figure 3, Figure 4, and Figure 5).3,9,17,23 The pool of fibrogenic pancreatic stellate cells has classically included native fibroblasts, circulating stem cells, epithelial-acinar mesenchymal transition cells, and now the proposed adult mesenchymal stem cell–like pluripotent pericyte.
The potential role of the pericyte as a pancreatic stellate cell-myofibroblast precursor in pancreatic fibrosis is supported by a number of studies, which have highlighted the in vivo capacity of the pericyte to act as a mesenchymal precursor cell.19,24–33 These studies include liver and renal fibrosis, in which resident pericyte cells have been shown to differentiate into myofibroblasts.24,29
The findings of IEI pericyte hypercellularity (due to hyperplasia and/or migration) and the close association with early fibrillar-banded collagen in the db/db diabetic model and Ren23,4 and HIP9 rodent models of T2DM suggest that the pericyte is playing an important role in IEI fibrosis. In addition to being supportive mural cells of the capillary endothelium and their important role in connecting capillaries throughout the endocrine and exocrine pancreas, our recent research and observational findings presented herein strongly suggest that pericytes may be the pancreatic stellate cell.3,9
These recent findings further demonstrate that intra-islet, IEI, endoacinar, and interlobular periacinar interstitial fibrosis appear to originate around capillaries in the islet (pericapillary fibrosis) (Figure 2C), IEI, and the triad of small arteries, veins, and ducts within the interstitium. This early perivascular-periductal fibrosis appears to emanate from these regions and involve the remainder of the loose areolar interstitial matrix, and it progresses to remaining regions. These findings also suggest that early fibrosis in the interstitium contemporaneously involves both vascular and ductal structures. Of importance, these findings support our previously held notion that the mural pericyte may be the linchpin linking vascular oxidative stress to interstitial-organ fibrosis and pancreatic failure.3
Insulo-Acinar-Ductal-Portal Vascular Pathway and Insulo-Acinar-Ductal-Pancreatic Enzyme–Incretin Gut Hormone Axis Communications Are Lost
The insulo-acinar-ductal-portal vascular pathway has been known and verified by matrix reconstruction studies for almost 3 decades.34,35 The microcirculation of the endocrine and exocrine pancreas travels through the continuous loose areolar matrix of the interstitial regions. Acini secrete their digestive pancreatic enzymes (PEs) into a central lumen lined by centroacinar cells. These small intercalated ducts drain into the intralobular, interlobular, and main/accessory pancreatic ducts within the interstitium to deliver their PE contents to the gastrointestinal tract (gut) to aid in the digestion of oral nutrients (fat, carbohydrates, and proteins). Properly digested nutrients are capable of activating the enteroendocrine L-cells of the ileum and colon to synthesize and secrete one of the incretins, glucagon-like peptide-1 (GLP-1).
The matrix connections are the critical structural pathways for each of these conduit systems, and interstitial fibrosis has a detrimental effect not only on the microcirculation but also on the ductal system, resulting in impaired insulin delivery to the hepatic portal system and impaired delivery of PE to the gut.
When pancreatic endocrine-exocrine tissue was viewed with low-magnification TEM, the less electron-dense matrix of the endoacinar and interlobular periacinar interstitium appeared similar to a road map, which we now term the pancreatic superhighway (Figure 3). Further, the matrix road map analogy allows envisioning fibrosis as a roadblock to the pancreatic superhighway that impairs cellular-molecular transportation and results in lost communication (Figure 6).
Clinical Implications of These Findings
Pericapillary fibrosis and islet amyloid deposition in the islet capillaries and efferent islet venules could certainly interfere with the delivery of insulin and result in a delay of first-phase insulin secretion in T2DM.36 Likewise, exocrine pancreatic periductal fibrosis could interfere with the delivery of PE to the gut, with the proper digestion of orally ingested nutrients, and with the microcirculation of the pancreas. Therefore, interstitial fibrosis may result in the loss of not only the endocrine effects associated with the microcirculation but also the cross-talk paracrine effects that islet, acinar, and ductal cells share.2
The Incretin Effect: GLP-1
As interstitial fibrosis progresses, there is increasing impairment of insulo-acinar-portal vascular pathway (microcirculation) and insulo-acinar-ductal-pancreatic enzyme–incretin gut hormone axis (ductal system) transportation of cells and molecules (islet hormones including insulin and PE) and paracrine signaling communications. As a result, islet hormones, including insulin’s trophic effects on acinar and ductal cells, may be impaired or lost. Also, the roadblock to the pancreatic superhighway may impair PE secretion to the gut and interfere with the proper digestion of oral nutrients and may decrease the stimulatory effect on the enteroendocrine L-cells in the ileum and colon to synthesize and secrete GLP-1 (Figure 7). Of interest, GLP-1 is known to be decreased in prediabetes and overt T2DM in humans,37,38 and recently, investigators have demonstrated an increase in GLP-1, plasma insulin, plasma C-peptide, and total insulin following PE replacement therapy. These findings suggest that the secretion of GLP-1 is under the influence of the proper digestion and absorption of nutrients in the small bowel and that PE supplementation increases GLP-1 and insulin secretion.39
The “incretin effect” is a term that has persisted in the literature for over 4 decades and was initially used to describe the finding that an oral glucose load produces a greater insulin response than an intravenous glucose infusion resulting in an equivalent glucose rise to the orally ingested glucose.40,41 Since only the incretin hormone GLP-1 and not the glucose-dependent insulinotropic hormone (GIP) have been found to be reduced in T2DM patients following an oral glucose load, we have focused our attention on GLP-1.42,43 Further, GIP does not stimulate insulin secretion in T2DM patients in physiologic or pharmacologic doses.43
There are multiple defects in insulin’s secretion and signaling in T2DM; however, the reduced or absent incretin effect (contributing between 25% and 60% to the insulin secretory response following an oral glucose challenge43) has only recently become of clinical importance due to the availability of GLP-1 mimetics and dipeptidyl peptidase IV (DPP-IV) inhibitor therapies. These newer treatment options (incretin mimetics and DPP-IV inhibitors) have expanded the treatment modalities, based on the early findings of GLP-1 being reduced in prediabetes and overt T2DM.36,37 Of importance, these newer therapies for patients with T2DM have resulted in an increased quality of life and have allowed hemoglobin A1c goals to be reached in many patients with T2DM.
Concurrent with our recent findings, there were publications that demonstrated the presence of impaired pancreatic exocrine function in T2DM as measured by fecal elastase-1 levels.44–46 Fecal elastase-1 levels (normal, >200 μg/g) were decreased to <100 μg/g in patients with T2DM (12%–20%) and type 1 diabetes mellitus (26%–44%),44 while a different study found that fecal elastase-1 levels were decreased in 28% of the T2DM population.46 It is important that these data support the association of T2DM with exocrine insufficiency and pancreatic diabetes or type 3c diabetes mellitus.44,45
These observational studies of the db/db diabetic obese mouse model coupled with findings in the Ren2, HIP, and ZDF rodent models demonstrate structural changes that likely reduce pancreatic endocrine-exocrine communication and function. Structural changes include a widening and hypercellularity consisting primarily of pericytes and occasional inflammatory cells within the IEI. In addition, the pericyte is associated with the early formation of organized banded fibrillar collagen of early fibrosis. The pancreatic matrix is continuous and appears similar to a road map, with the matrix representing a pancreatic superhighway. It is known that fibrosis of the IEI and pancreatic interstitium develops as these animal models age and as T2DM progresses in humans (Figure 6).
The pancreas is connected by a continuous matrix supporting communication and transportation of molecules and cells. Interstitial matrix remodeling fibrosis creates a roadblock to this continuous pancreatic superhighway and results in lost connections and communication. The images presented make it obvious that we are dealing with a continuously integrated organ, in which endocrine and exocrine glands are structurally and functionally interconnected with a continuous matrix supporting the microcirculation and ductal function, allowing for a well-orchestrated functional response.
GLP-1 mimetics and DPP-IV inhibitor therapies act to bypass these fibrotic roadblocks, and if we can decrease the fibrotic roadblocks by decreasing the multiple metabolic toxicities, including glucotoxicity and oxidative stress, and restoring the incretin effect earlier in the impaired glucose tolerance/prediabetes phase, we may be able to delay the natural progressive nature of T2DM. No longer can we assume that diarrhea in diabetes is due to diabetic neuropathy unless pancreatic insufficiency is shown not to be present.
We suggest the use of PE supplementation in patients who have demonstrated pancreatic insufficiency and further suggest that early PE supplementation may have a preventive effect on the natural progressive history of T2DM in patients with impaired glucose tolerance/prediabetes.
Disclosures: This research was supported by the National Institutes of Health (R01 HL73101-01A1), the Veterans Affairs Merit System (0018), Novartis Pharmaceuticals (JRS), and the Missouri Kidney Program and Veterans Affairs VISN 15 (AW-C).