Wan-Chun Li

β-Cell Growth and Regeneration: Replication is Only Part of the Story

> “I am accordingly of the opinion that the normal regulation of islet content in the pancreas is by interstitial growth of pre-existing islets and by the formation of new islets from the duct epithelium, and not at all by the formation of new islets out of acini.” > > R.R. Bensley Am J Anatomy 1911;12:297–388 For almost a century (for historical review, see 1) both β-cell replication and neogenesis (the differentiation of new islet cells from progenitors or stem cells) have been thought to be responsible for postnatal growth of the endocrine pancreas. Even though doubts have been raised in recent years about the existence and importance of neogenesis, this skepticism may be subsiding. Replication and neogenesis are not mutually exclusive. Both processes often occur simultaneously, as seen during the regeneration that follows pancreatic injury. However, there are important differences in the balance of these two pathways that depend upon species and age. Replication of β-cells is an important mechanism particularly in adult rodents, but there are compelling data that after-birth progenitors also have a role in renewal and growth of islets. Eventually one or both of these pathways may be manipulated for therapeutic treatment of diabetes. Since we and others have extensively reviewed the regulation of β-cell mass (2,3), this Perspective will specifically address the contributions of the neogeneic pathway to new β-cell formation, considering whether postnatal neogenesis occurs, to what extent; possible differences between mammalian species; and whether it might be exploited therapeutically. ### β-Cell expansion in normal growth. The concept that β-cell mass is dynamic and increases and decreases both in function and mass to maintain the glycemic level within a very narrow physiological range (4) is now generally accepted. In both normal and pathophysiological states, the mechanisms responsible are changes in replication and neogenesis, changes in individual cell volume, and changes in cell loss or death rates. In …

Transdifferentiation of pancreatic ductal cells to endocrine β-cells

The regenerative process in the pancreas is of particular interest, since diabetes, whether Type 1 or Type 2, results from an inadequate amount of insulin-producing beta-cells. Islet neogenesis, or the formation of new islets, seen as budding of hormone-positive cells from the ductal epithelium, has long been considered to be one of the mechanisms of normal islet growth after birth and in regeneration, and suggested the presence of pancreatic stem cells. Results from the rat regeneration model of partial pancreatectomy led us to hypothesize that differentiated pancreatic ductal cells were the pancreatic progenitors after birth, and that with replication they regressed to a less differentiated phenotype and then could differentiate to form new acini and islets. There are numerous supportive results for this hypothesis of neogenesis, including the ability of purified primary human ducts to form insulin-positive cells budding from ducts. However, to rigorously test this hypothesis, we took a direct approach of genetically marking ductal cells using CAII (carbonic anhydrase II) as a duct-cell-specific promoter to drive Cre recombinase in lineage-tracing experiments using the Cre-Lox system. We show that CAII-expressing pancreatic cells act as progenitors that give rise to both new islets and acini after birth and after injury (ductal ligation). This identification of a differentiated pancreatic cell type as an in vivo progenitor for all differentiated pancreatic cell types has implications for a potential expandable source for new islets for replenishment therapy for diabetes either in vivo or ex vivo.

Activation of pancreatic-duct-derived progenitor cells during pancreas regeneration in adult rats

The adult pancreas has considerable capacity to regenerate in response to injury. We hypothesized that after partial pancreatectomy (Px) in adult rats, pancreatic-duct cells serve as a source of regeneration by undergoing a reproducible dedifferentiation and redifferentiation. We support this hypothesis by the detection of an early loss of the ductal differentiation marker Hnf6 in the mature ducts, followed by the transient appearance of areas composed of proliferating ductules, called foci of regeneration, which subsequently form new pancreatic lobes. In young foci, ductules express markers of the embryonic pancreatic epithelium – Pdx1, Tcf2 and Sox9 – suggesting that these cells act as progenitors of the regenerating pancreas. The endocrine-lineage-specific transcription factor Neurogenin3, which is found in the developing embryonic pancreas, was transiently detected in the foci. Islets in foci initially resemble embryonic islets in their lack of MafA expression and lower percentage of β-cells, but with increasing maturation have increasing numbers of MafA+ insulin+ cells. Taken together, we provide a mechanism by which adult pancreatic duct cells recapitulate aspects of embryonic pancreas differentiation in response to injury, and contribute to regeneration of the pancreas. This mechanism of regeneration relies mainly on the plasticity of the differentiated cells within the pancreas.

Isolation and Culture of Adult Mouse Hepatocytes

The liver performs a multitude of functions including the regulation of carbohydrate, fat, and protein metabolism, the detoxification of endo- and xenobiotics, and the synthesis and secretion of plasma proteins and bile. Isolated hepatocytes constitute a useful technique for studying liver function in an in vitro setting. Here we describe a method for the isolation of hepatocytes from adult mouse liver. The principle of the method is the two-step collagenase perfusion technique which involves sequential perfusion of the liver with ethylenediaminetetraacetic acid and collagenase. Following isolation, the cells can either be used for short-term studies or, alternatively, maintained in culture for prolonged periods to study long-term changes in gene expression. The protocol for mouse hepatocyte isolation may be applied to both normal and transgenic mice.

In vitro transdifferentiation of hepatoma cells into functional pancreatic cells

We have characterised the transdifferentiation of human HepG2 (hepatoma) cells to pancreatic cells following introduction of an activated version of the pancreatic transcription factor Pdx1 (XlHbox8-VP16). The following questions are addressed: (1) are all types of pancreatic cells produced? (2) is the requirement for expression of the transgene temporary or permanent? (3) are the transdifferentiated beta-cells responsive to physiological stimuli? The results showed that both pancreatic exocrine cells (by detection of amylase protein), and endocrine cells (by detecting insulin, glucagon and somatostatin proteins) are induced after XlHbox8VP16 transfection. Moreover, the hepatic phenotype becomes suppressed during transdifferentiation of hepatocytes to pancreatic cells. Requirement for the transgene is only temporary and it is no longer required once the pancreatic differentiation program is activated. Finally, we provided results to suggest that the transdifferentiated cells are functional by detecting: (1) functional markers for pancreatic beta-cells including prohormone convertase 1/3 (PC1/3), insulin C-peptide and glucagon-like peptide 1 receptor (GLP-1R), (2) increased insulin mRNA expression after treatment of cells with GLP-1 and betacellulin, physiological stimuli that regulate pancreatic function and (3) elevated insulin secretion after glucose challenge. The transdifferentiation of hepatic to pancreatic cells represents one possible source of beta-cells for human islet transplantation and this study shows that such a transdifferentiation can be achieved in vitro.

The molecular basis of transdifferentiation

There is now excellent experimental evidence demonstrating the remarkable ability of some differentiated cells to convert to a completely different phenotype. The conversion of one cellular phenotype to another is referred to as ‘transdifferentiation’ and belongs to a wider class of cell‐type switches termed ‘metaplasias’. Defining the molecular steps in transdifferentiation will help us to understand the developmental biology of the cells that interconvert, as well as help identify key regulatory transcription factors that may be important for the reprogramming of stem cells. Ultimately, being able to produce cells at will offers a compelling new approach to therapeutic transplantation and therefore the treatment and cure of diseases such as diabetes.

Islet neogenesis: a possible pathway for beta-cell replenishment

Diabetes, particularly type 1 diabetes, results from the lack of pancreatic β-cells. β-cell replenishment can functionally reverse diabetes, but two critical challenges face the field: 1. protection of the new β-cells from autoimmunity and allorejection, and 2. development of β-cells that are readily available and reliably functional. This chapter will examine the potential of endogenous replenishment of pancreatic β-cells as a possible therapeutic tool if autoimmunity could be blunted. Two pathways for endogenous replenishment exist in the pancreas: replication and neogenesis, defined as the formation of new islet cells from pancreatic progenitor/stem cells. These pathways of β-cell expansion are not mutually exclusive and both occur in embryonic development, in postnatal growth, and in response to some injuries. Since the β-cell population is dramatically reduced in the pancreas of type 1 diabetes patients, with only a small fraction of the β-cells surviving years after onset, replication of preexisting β-cells would not be a reasonable start for replenishment. However, induction of neogenesis could provide a starting population that could be further expanded by replication. It is widely accepted that neogenesis occurs in the initial embryonic formation of the endocrine pancreas, but its occurrence anytime after birth has become controversial because of discordant data from lineage tracing experiments. However, the concept was built upon many observations from different models and species over many years. Herein, we discuss the role of neogenesis in normal growth and regeneration, as learned from rodent models, followed by an analysis of what has been found in humans.

Differential microRNA Profiles Predict Diabetic Nephropathy Progression in Taiwan.

OBJECTIVES Diabetic nephropathy (DN) is a major leading cause of kidney failure. Recent studies showed that serological microRNAs (miRs) could be utilized as biomarkers to identify disease pathogenesis; the DN-related miRs, however, remained to be explored. METHODS A prospective case-control study was conducted. The clinical significance of five potential miRs (miR-21, miR-29a, miR-29b, miR-29c and miR192) in type 2 Diabetes Mellitus (T2DM) patients who have existing diabetic retinopathy with differential Albumin:Creatinine Ratio (ACR) and estimated Glomerular Filtration Rate (eGFR) was performed using quantitative RT-PCR analysis. The subjects with diabetic retinopathy enrolled in Taipei City Hospital, Taiwan, were classified into groups of normal albuminuria (ACR<30mg/g; N=12); microalbuminuria (30mg/g300mg/g; N=21) as well as 18 low-eGFR (eGFR<60ml/min) and 32 high-eGFR (eGFR>60ml/min). The level of serum miRs was statistically correlated with age, Glucose AC, ACR, eGFR and DN progression. RESULTS The levels of miR-21, miR-29a and miR-192 were significantly enriched in the overt proteinuria group compared with microalbuminuria and/or overt proteinuria groups. It was shown that only miR-21 level was significantly up-regulated in low-eGFR group compared with high-eGFR patients. Interestingly, Pearsons correlation coefficient analysis demonstrated that DN progressors showed significantly greater levels of miR-21, miR-29a, miR-29b and miR-29c in comparison with non-progressors implying the clinical potential of DN associated miRs in monitoring and preventing disease advancement. CONCLUSION Our findings showed that miR-21, miR-29a/b/c and miR-192 could reflect DN pathogenesis and serve as biomarkers during DN progression.

Circulating microRNA as a diagnostic marker in populations with type 2 diabetes mellitus and diabetic complications

Abstract Diabetes mellitus (DM) is a global health care issue resulting from hyperglycemia‐mediated life‐threatening complications. Although the use of glucose‐lowering agents is routinely practiced, high dependence on medication leads to poor quality of life for DM patients. While it is still not feasible to precisely determine the critical timing when DM is truly established, perhaps the best way to reduce DM‐associated mortality is to prevent it. To this end, an exploration of prognostic molecules sensitive enough to detect early physiological alteration at the initiating stage would be required. Recently discovered small noncoding molecules, microRNAs (miRs), in body fluid seem promising to be utilized as a biomarker to monitor DM initiation and progression, as it is believed that expression of circulating miRs reflects disease pathology. Current DM‐related miRs were often referred to miRs differentially expressed in insulin target organs (liver, muscle, and adipose tissues) or circulating blood (peripheral blood) in diabetic patients compared to their control counterparts, although these miRs could merely be resultant nucleotides from DM‐induced organ impairment instead of the indicators of onset/progression of DM. In the current review, studies showing circulating miRs associated with type 2 DM and its complications are summarized, and future scope of using miRs as biomarkers for disease prognosis/diagnosis is also emphasized.

The regulation and function of the NUAK family

AMP-activated protein kinase (AMPK) is a critical regulator of cellular and whole-body energy homeostasis. Twelve AMPK-related kinases (ARKs; BRSK1, BRSK2, NUAK1, NUAK2, QIK, QSK, SIK, MARK1, MARK2, MARK3, MARK4, and MELK) have been identified recently. These kinases show a similar structural organization, including an N-terminal catalytic domain, followed by a ubiquitin-associated domain and a C-terminal spacer sequence, which in some cases also contains a kinase-associated domain 1. Eleven of the ARKs are phosphorylated and activated by the master upstream kinase liver kinase B1. However, most of these ARKs are largely unknown, and the NUAK family seems to have different regulations and functions. This review contains a brief discussion of the NUAK family including the specific characteristics of NUAK1 and NUAK2.