Solid organ transplantation is currently an important

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1 REVIEW Post-Transplant Diabetes Mellitus: Causes, Treatment, and Impact on Outcomes Vijay Shivaswamy, Brian Boerner, and Jennifer Larsen Division of Diabetes, Endocrinology, and Metabolism (V.S., B.B., J.L.), Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska 68198; and VA Nebraska-Western Iowa Health Care System (V.S.), Omaha, Nebraska Post-transplant diabetes mellitus (PTDM) is a frequent consequence of solid organ transplantation. PTDM has been associated with greater mortality and increased infections in different transplant groups using different diagnostic criteria. An international consensus panel recommended a consistent set of guidelines in 2003 based on American Diabetes Association glucose criteria but did not exclude the immediate post-transplant hospitalization when many patients receive large doses of corticosteroids. Greater glucose monitoring during all hospitalizations has revealed significant glucose intolerance in the majority of recipients immediately after transplant. As a result, the international consensus panel reviewed its earlier guidelines and recommended delaying screening and diagnosis of PTDM until the recipient is on stable doses of immunosuppression after discharge from initial transplant hospitalization. The group cautioned that whereas hemoglobin A1C has been adopted as a diagnostic criterion by many, it is not reliable as the sole diabetes screening method during the first year after transplant. Risk factors for PTDM include many of the immunosuppressant medications themselves as well as those for type 2 diabetes. The provider managing diabetes and associated dyslipidemia and hypertension after transplant must be careful of the greater risk for drug-drug interactions and infections with immunosuppressant medications. Treatment goals and therapies must consider the greater risk for fluctuating and reduced kidney function, which can cause hypoglycemia. Research is actively focused on strategies to prevent PTDM, but until strategies are found, it is imperative that immunosuppression regimens are chosen based on their evidence to prolong graft survival, not to avoid PTDM. (Endocrine Reviews 37: 37 61, 2016) I. Introduction II. Diagnosis and Incidence III. Known and Potential Risk Factors A. Pre-existing diabetes risk B. Associated candidate genes C. Role of immunosuppression agents D. Potential role of stress, inflammation, and infection E. Potential role of vitamin D or other factors IV. The Impact of PTDM on Transplant Outcomes A. Introduction B. Kidney transplant outcomes C. Outcomes after other organ transplant groups V. Treating Diabetes After Transplant A. Treatment of the hospitalized patient B. Outpatient glucose management C. Preventing cardiovascular disease D. Eye care, foot care, preventing infections, and reproductive health E. Importance of team-based care VI. Prevention of PTDM VII. Summary ISSN Print X ISSN Online Printed in USA Copyright 2016 by the Endocrine Society Received July 31, Accepted November 24, First Published Online December 9, 2015 I. Introduction Solid organ transplantation is currently an important option for the treatment of many types of organ failure, including kidney, liver, heart, pancreas, lung, and small bowel. The introduction of cyclosporine heralded a new era in transplant outcomes, and outcomes have continued to improve with new regimens and improvements in care. Immunosuppression has so improved graft and patient survival after kidney transplant that the number one cause of graft failure is patient death of other causes unrelated to graft failure (1, 2). Kidney transplant improves patient survival and is also more cost-effective than dialysis. All of these improvements have, in turn, led to more individuals receiving solid organ transplants (Figure 1) who require long-term care. Abbreviations: BMI, body mass index; CF, cystic fibrosis; CFRD, CF-related diabetes; CMV, cytomegalovirus; CNI, calcineurin inhibitor; DPP-4, dipeptidyl-peptidase-4; GLP-1, glucagonlike peptide-1; HbA1c, hemoglobin A1c; HCV, hepatitis C virus; IFG, impaired fasting glucose; IRS, insulin receptor substrate; mtor, mammalian target of rapamycin; mtorc, mtor-containing complex; NODAT, new-onset diabetes after transplantation; OGTT, oral glucose tolerance test; PCKD, polycystic kidney disease; PI3K, phosphatidylinositol 3 kinase; PTDM, post-transplant diabetes mellitus; SNP, single nucleotide polymorphism; SPK, simultaneous pancreas-kidney; TPN, total parenteral nutrition. doi: /er Endocrine Reviews, February 2016, 37(1):37 61 press.endocrine.org/journal/edrv 37

2 38 Shivaswamy et al Post-Transplant Diabetes Mellitus Endocrine Reviews, February 2016, 37(1):37 61 Figure 1. Figure 1. Total solid organ transplants performed in the United States. Numbers represent transplants performed from January 1988 to March 31, 2015, and reported to the United Network for Organ Sharing. There was great hope that new steroid-free immunosuppression regimens would significantly reduce many side effects, including diabetes risk, but, in fact, even without corticosteroids, risk of diabetes remains a concern. In fact, the increasing frequency of obesity, particularly in kidney transplant candidates, has also increased the risk of post-transplant diabetes mellitus (PTDM). This review will discuss the diagnosis of PTDM, based on the latest consensus guidelines, and how different screening practices and guidelines in the past affect our knowledge of the epidemiology of PTDM and perhaps the reported consequences of PTDM. We will discuss the factors that contribute to PTDM, including genetics, family traits, the prescribed immunosuppressants themselves, the potential role of inflammation, and factors yet to be fully proven. Treatment of any type of diabetes can be challenging after transplant, whetherpre-existingdiabetesorptdm. We will describe best practices for glucose management in the hospital and review the small studies of type 2 diabetes treatment agents that have assessed their safety in transplant recipients and other considerations for management of diabetes in an outpatient setting. Finally, we will discuss what approaches are being taken to prevent or reduce PTDM or its impact on outcomes of transplant recipients. II. Diagnosis and Incidence In 2003, the first International Consensus Guidelines for new-onset diabetes after transplantation (NODAT) were published(3). Although these criteria were focused on the diagnosis of diabetes after kidney transplant, they have been largely adopted by other transplant groups. Based on American Diabetes Association and World Health Organization (WHO) criteria for nontransplant patients at that time, diagnosis of NODAT could result from a fasting glucose 126 mg/dl (7 mmol/l) on more than one occasion, random glucose 200 mg/dl (11.1 mmol/l) with symptoms, or a 2-hour glucose level after a 75-g oral glucose tolerance test (OGTT) of 200 mg/dl (11.1 mmol/l) (4, 5). In October 2013, a second international consensus panel met to update criteria and other data regarding NODAT and to evaluate the addition of hemoglobin A1C as a criterion, as it had been defined by the American Diabetes Association in 2010 in nontransplant adults (4, 5). The summary of their discussion and major recommendations were published in 2014 (see Table 1; Ref. 6). Table 1. Diagnosis of PTDM Based on recent International Consensus Guidelines (6), the diagnosis of PTDM can be made using any of the following American Diabetes Association/World Health Organization criteria for the diagnosis of diabetes (4, 5) once the transplant recipient has been discharged from the hospital and tapered to their maintenance immunosuppression. However, HbA1c should not be used alone to screen for diagnosis of PTDM within the first year after transplant. 1) Fasting glucose 126 mg/dl (7 mmol/l) on more than one occasion. 2) Random glucose 200 mg/dl (11.1 mmol/l) with symptoms. 3) Two-hour glucose after a 75-g OGTT of 200 mg/dl (11.1 mmol/l). 4) HbA1c 6.5%.

3 doi: /er press.endocrine.org/journal/edrv 39 The first recommendation of the group was to change the name from NODAT to PTDM because many cases of diabetes first identified after transplant are likely not new. There is considerable variation among transplant centers as to how or if candidates are screened for diabetes before transplant, but most centers use only fasting glucose and hemoglobin A1C for screening candidates. These methods are less sensitive than the OGTT for identifying diabetes in patients with chronic renal failure (7). Thus, it is unknown how many of those first recognized as having diabetes after transplant are truly new. Second, the timing of the PTDM diagnosis was re-evaluated. The original criteria did not exclude diagnosis of NODAT from the immediate postoperative hospitalization when many recipients receive large doses of corticosteroids. With greater inpatient glucose screening, there is increasing awareness of the number of transplant recipients who have glucose intolerance or could be diagnosed with PTDM during this immediate post-transplant period (8, 9). Because the hyperglycemia does not always persist after discharge and the diagnosis of diabetes in most nontransplant populations is generally reserved for the outpatient setting, the consensus panel recommended delaying the evaluation for and diagnosis of PTDM until the recipient had been discharged from the hospital, was stable, and had tapered to likely chronic immunosuppression doses. The third major recommendation of the consensus panel concerned the use of hemoglobin A1C for diagnosis of PTDM. For many reasons, including reduced red blood cell survival after transplant, hemoglobin A1C is a less reliable measure for identifying significant glucose intolerance in the first 12 months after transplant (10, 11). The panel concluded that whereas hemoglobin A1C can be used to diagnose diabetes if elevated ( 6.5%), it should not be used alone as a screen for PTDM, particularly in the first year after transplant. It should be noted that, currently, the diagnosis of PTDM has no end date. If an adult or a pediatric solid organ transplant recipient is diagnosed with diabetes 1, 10, or 40 years later, it is still eligible to be called PTDM. Thus, even with the best of strategies to prevent the immediate onset of significant hyperglycemia, glucose intolerance related to age cannot be avoided, and the significance or impact of the diagnosis of PTDM made in the first year vs five years after transplant has yet to be determined. Incidence varies with the type of transplant because of differences in age, body mass index (BMI), and underlying risk of the candidate group. Incidence also varies among transplant centers because of variations in the patients they serve with respect to race/ethnicity or prevalence of obesity, which affect risk, as well as variations in their usual immunosuppression regimen. However, the data published before the first consensus conference were quite variable, many centers did not fully embrace those first criteria, and no data have been published since the second criteria published in Thus, whereas we have summarized the incidence of PTDM published in Table 2, these should be considered relative, not absolute, risk rates because those rates are likely to be significantly changed from what we and others have published in the past once the new criteria for diagnosis of PTDM are implemented (9, 12 16). III. Known and Potential Risk Factors A. Pre-existing diabetes risk Just as with type 2 diabetes, PTDM is more likely to occur in individuals with pre-existing type 2 diabetes risk factors, including age and a family history of type 2 diabetes, and in those with race or ethnicity considered at higher risk than Caucasians for type 2 diabetes particularly African Americans and Hispanics (16) (Figure 2). Although Native Americans are among those with the highest risk for type 2 diabetes, there are less data identifying them at high risk for PTDM because most Native Americans presenting for transplant have already been diagnosed with diabetes. Importantly, obesity, whether defined as elevated BMI or increased waist circumference, has also been associated with increased risk of type 2 diabetes as well as PTDM. Obesity can predate the transplant, but weight gain after transplant can also contribute. B. Associated candidate genes Multiple studies have found an association between type 1 and type 2 diabetes candidate gene single nucleotide polymorphisms (SNPs) with risk for PTDM, including several genes implicated in maturity onset diabetes of the young syndromes. For example, in Hispanic kidney transplant recipients, SNPs in insulin receptor substrate (IRS)-1 Table 2. Incidence of PTDM as Described After 2003 International Consensus Guidelines (3) Type of Transplant Incidence, % Refs. Kidney , 13, 15, 43, 126, 211 Heart Liver Lung Studies of incidence published after the first International Consensus Guidelines are provided. However, even these have not always described their methods of screening for diabetes before or after transplant. Because there are no studies yet published after the more recent international guidelines, which excludes hyperglycemia that occurs in the immediate post-transplant hospitalization and follow-up, the overall incidence could change for kidney transplant, in particular. There are too few studies reported after lung transplant to report incidence.

4 40 Shivaswamy et al Post-Transplant Diabetes Mellitus Endocrine Reviews, February 2016, 37(1):37 61 Figure 2. Figure 2. Potential contributors to PTDM. Risk factors for PTDM including pre-existing diabetes risk, immunosuppressant agents used for treatment, inflammation, and other factors. FH, family history; DM, diabetes; HDL, high-density lipoprotein; TX, transplant; Vit D, vitamin D; Mg, magnesium; HepC, hepatitis C. and hepatocyte nuclear factor 4 were associated with diabetes after kidney transplant (17). Additional type 2 candidate gene SNPs associated with PTDM include TCF7L2, KCNJ11-Kir6.2, and some but not all variants of KCNQ1 (18 22). Those carrying multiple SNPs in diabetic genes were at even greater risk. PTDM has also been associated with SNPs in multiple IL genes, particularly IL-2, IL-7R, IL-17R, IL-1B, IL-4, IL-17-RE, IL-17R, and IL-17RB (23). Finally, SNPs in the transcription factor NFATc4 and adiponectin are also associated with PTDM (24, 25). One of the commonly used classes of immunosuppressant agents, the calcineurin inhibitors (CNIs; cyclosporine and tacrolimus), bind to nuclear factor of activated T-cells (NFAT). NFAT, in turn, can down-regulate adiponectin transcription, so individuals with SNP in this candidate gene may be at greater risk for glucose intolerance related to CNIcontaining regimens. Some genes associated with risk of end-organ failure leading to solid organ transplant are also associated with risk of PTDM. Specifically, several retrospective studies have identified autosomal dominant polycystic kidney disease (PCKD) as increasing the risk of PTDM after kidney transplant (13, 26 28). However, at least one retrospective cohort study, where the diabetes screening protocol was not well described, showed no significant difference in new-onset diabetes from matched controls (29). Yet several studies have suggested a potential mechanism for risk. In one study, carriers of the C variant gene polymorphism, among those with autosomal dominant PCKD, were found to have higher body fat content and glucose response to oral glucose challenge, but no insulin secretory defect (30). One of the candidate genes for type 2 diabetes and PTDM, hepatocyte nuclear factor 4, has also been identified as a disease modifier in an established PCKD animal model, resulting in greater cystic changes in the kidney (31). Thus, individuals with this SNP in addition to autosomal dominant PCKD may need transplant earlier and may be at greater risk for diabetes as well. Cystic fibrosis (CF) is a genetic disease that can lead to the need for lung transplant. CF is also associated with a distinct type of diabetes, CFrelated diabetes (CFRD). Thus, it is not surprising that those individuals with CF who have not developed CFRD before transplant are at higher risk for developing diabetes after lung transplant than other lung transplant candidates (32). Although some transplant groups refer to this as post-transplant diabetes, it might also be considered CFRD, for which this group is also at risk. Which designation should be used has not been discussed by any consensus panel or transplant organization. C. Role of immunosuppression agents Corticosteroids are well established to cause hyperglycemia through several mechanisms: by inducing or worsening pre-existing insulin resistance, increasing hepatic gluconeogenesis, and long-term, by stimulating appetite and weight gain (33 35). The impact is dose-dependent. High-dose corticosteroids, often used as part of induction protocols in the immediate post-transplant hospitalization, have much greater impact than chronic low-dose corticosteroids that are common to many maintenance immunosuppression protocols. A recent prospective randomized trial of early withdrawal of corticosteroids vs remaining on low-dose chronic prednisone (5 mg/d) from 6 months to 5 years after kidney transplant

5 doi: /er press.endocrine.org/journal/edrv 41 showed that incidence of PTDM was minimally impacted (36). The remaining available immunosuppressive agents vary with respect to risk for PTDM. Mycophenolate mofetil and azathioprine have not been shown to have a large impact on insulin action or glucose metabolism and so do not appear to have a major role in PTDM. There is increasing evidence that the other commonly used immunosuppressants, particularly CNIs (eg, tacrolimus and cyclosporine) and inhibitors of the mammalian target of rapamycin (mtor; eg, sirolimus or rapamycin and everolimus), may contribute to PTDM. Our work in animals suggests a dose-dependent effect of both tacrolimus and sirolimus on glucose metabolism (37), although there are no comparable human studies. However, there are reports that both glucose intolerance and the dyslipidemia that occurs predominantly with mtor inhibitors improve as the dose is reduced. Cyclosporine was the first CNI to be introduced, and it greatly improved graft survival with lower doses of corticosteroids. Because lower doses of corticosteroids were needed, both to prevent and treat rejection, less hyperglycemia was observed, initially leading many to believe these agents had no impact on insulin secretion or action. It wasn t until tacrolimus was introduced that the association between CNIs and glucose intolerance was first identified (38, 39). Tacrolimus therapy increased diabetes risk in transplant recipients already known to be at highest risk, such as African Americans and those with prior history of hepatitis C (40, 41). Although tacrolimus appeared to increase relative risk for developing PTDM more than cyclosporine treatment, it became apparent that cyclosporine was also associated with risk, 18% compared to 8%, respectively, based on data from the U.S. Renal Database for the 2-year incidence of PTDM after kidney transplant (42). A greater relative risk of tacrolimus compared to cyclosporine was also confirmed by a randomized controlled trial (43). Tacrolimus was also shown to increase the incidence of prediabetes after kidney transplant (33% at 12 mo) (44). It should be noted that not all published studies have shown that tacrolimus and/or cyclosporine significantly affects glucose intolerance. Shihab et al (45) showed that switching from cyclosporine to tacrolimus resulted in improved renal function and no increase in new-onset diabetes or hyperglycemia. However, this study defined diabetes in a way that may have underestimated PTDM and was inconsistent with the published 2003 International Consensus Guidelines; specifically, they defined diabetes patients as those who had a fasting glucose 140 mg/dl and required insulin treatment for more than 30 consecutive days, with anything less considered hyperglycemia. Robertson et al (46) studied the impact of cyclosporine on glucose homeostasis in 19 nondiabetic multiple sclerosis patients, noted to have ideal BMI, who were randomized to receive cyclosporine or placebo for up to 1 year; they were unable to show any effect of the prescribed cyclosporine on pancreatic -cell function or iv glucose tolerance, although the doses used were less than normally prescribed early after transplant. The absence of an effect might suggest that pre-existing risk for diabetes may be additive because this population was not well described but was less likely to have a comparable BMI or other risk than a transplant population. A number of investigator groups have evaluated the mechanism by which CNIs, tacrolimus or cyclosporine, impact glucose metabolism and diabetes risk in vitro, in animal models, and in patients (14, 37, 47, 48). Multiple mechanisms have been implicated for CNI-associated PTDM (Figure 3). CNIs have been shown to impair insulin secretion in clinical studies (49 53). Animal studies confirmed that treatment with tacrolimus worsened glucose tolerance both in vivo and in vitro and in normal as well as insulin-resistant models, predominantly through decreasing insulin secretion (54, 55). We have shown that short-term tacrolimus treatment of normal male and female Sprague-Dawley rats caused hyperglycemia with mild hyperinsulinemia in response to glucose challenge in a dose-dependent manner, suggesting mild insulin resistance; with longer duration of treatment, hyperinsulinemia was no longer observed, suggesting loss of insulin secretion (37, 54). Tacrolimus was also shown to reduce -cell mass and increase islet apoptosis, but it had less impact on insulin signaling, suggesting that its predominant action was on insulin production (56). CNI treatment of animal models has also demonstrated decreased glucokinase activity and reduced insulin gene expression (57 59). Tacrolimus has also been shown to induce -cell apoptosis in both isolated rodent and human islets. Increased apoptosis was also observed after 24-hour treatment of isolated human islets with tacrolimus through the up-regulation of caspase-3 cleavage and activity (60). The apoptosis was associated with decreased Akt phosphorylation and IRS-2 mrna and protein levels, suggesting that CNIs impact human -cell survival in part through regulation of IRS-2 (61). Another study confirmed the effect of CNIs on abolishing glucose-induced IRS-2 mrna and protein levels in rat islet -cells (62). Pancreas allograft biopsies of pancreas transplant recipients have also demonstrated reversible cytoplasmic swelling and vacuolization of islets with tacrolimus and cyclosporine treatment (63). Although most of the clinical data have suggested that tacrolimus, in particular, has its predominant impact through reduced insulin secretion, more recent data sug-

6 42 Shivaswamy et al Post-Transplant Diabetes Mellitus Endocrine Reviews, February 2016, 37(1):37 61 Figure 3. Figure 3. Sirolimus and CNI impact on insulin signaling pathway. PDK1, phosphoinositide-dependent protein kinase 1; Rictor, rapamycin-insensitive companion of mtor; Proctor, protein observed with Rictor; Raptor, regulatory-associated protein of mtor; PRAS40, proline-rich Akt substrate 40; mlst8, mtor-associated protein, LST8 homolog; YY1, Yin Yang 1; msin1, mammalian stress-activated protein kinase interactions protein; p, phosphorylation. Note that direct stimulation is denoted by, inhibition is denoted by, and tentative inhibition by. gest that tacrolimus more than cyclosporine-based regimens can also increase insulin resistance after kidney transplant. This was first reported in animal studies but was also suggested in recent clinical studies as measured by homeostasis model assessment of insulin resistance and fasting insulin concentration (64 66). In human adipocytes, both cyclosporine and tacrolimus inhibited glucose uptake independent of insulin signaling by removing GLUT4 from the cell surface through an increased rate of endocytosis (67). In Wistar rats, cyclosporine reduced IRS-1 gene expression in liver and perirenal fat (68). The impact of CNI on insulin signal transduction may also be tissue-specific because cyclosporine treatment decreased IRS-1 protein in fat but not in liver (68). However, some human studies have not confirmed these results. In 10 healthy human volunteers with normal BMI ( kg/m 2 ), Øzbay et al (69) showed that an acute infusion of CNIs over 5 hours increased insulin sensitivity as determined by hyperinsulinemic-euglycemic clamp. A greater increase in sensitivity was observed with cyclosporine (25%) than tacrolimus (13%), but there was no change in first-phase insulin secretion or insulin pulsatility. Although the concentration achieved is comparable to goal doses after treatment, their BMI did not suggest that the impact of CNIs on glucose metabolism may require more than 5 hours or other additional susceptibility as with elevated BMI. Rickels et al (70) compared type 1 diabetic islet transplant recipients with nondiabetic kidney and portally drained simultaneous pancreas-kidney (SPK) recipients using glucose-potentiated arginine testing. Although islet transplant recipients had impaired insulin secretion, the acute insulin response in tacrolimus-treated SPK and nondiabetic kidney transplant recipients was similar to kidney transplant donors at 3 48 months (SPK) and 6 60 months (nondiabetic kidney) after transplant on maintenance immunosuppression. It should be noted that the BMI values were matched between the groups and were lower than most transplant recipients that develop PTDM: 24 1 kg/m 2 for the normal controls; 21 1 kg/m 2 for

7 doi: /er press.endocrine.org/journal/edrv 43 islet transplant recipients; 23 1 kg/m 2 for SPK; 23 1 kg/m 2 in nondiabetic kidney recipients; and 24 1 kg/m 2 for kidney donors. These studies do not exclude the potential impact of CNIs on insulin action but again suggest that recipients may not all be equally at risk. Cottrell (71) used OGTT and frequently sampled iv glucose tolerance testing with minimal model analysis to evaluate insulin sensitivity and -cell function over 48 months in a cross-sectional study of 33 SPK recipients treated with a cyclosporine-based regimen. In this cohort that was selected because they were euglycemic, -cell function was reduced at 3 6 months but improved by 12 months and normalized by months, maintaining euglycemia with greater insulin secretion. Again, the candidates were type 1 diabetes patients and were treated with both cyclosporine and corticosteroids, so it is difficult to isolate the potential impact of the cyclosporine from the corticosteroids or to compare the results to a nonselected, kidney-transplant population with PTDM. Additionally, in this group the pancreas graft was placed into the systemic circulation, which results in significant systemic hyperinsulinemia that makes the insulin concentrations harder to interpret. Finally, eight islet transplant recipients treated predominantly with tacrolimus and minimal corticosteroids were also compared to patients with type 1 diabetes and normal controls using a frequently sampled iv glucose tolerance test to evaluate insulin sensitivity, glucose effectiveness, and free fatty acid dynamics. Insulin sensitivity was greater in the islet transplant and normal controls compared to the type 1 diabetes subjects, suggesting again that at least in this small cohort of thin (BMI, kg/m 2 ) islet transplant recipients, the tacrolimus dose administered did not reduce insulin sensitivity (72). Another mechanism by which CNI has been suggested to contribute to hyperglycemia is hypomagnesemia (73, 74). Hypomagnesemia alone is known to impact insulin signaling (75), is commonly associated with CNI treatment, and is associated with increased risk for PTDM. In a retrospective analysis of 254 renal transplant recipients, patients who developed PTDM had lower serum magnesium levels in the first month after transplant compared to non-ptdm recipients, and those with the lowest magnesium levels developed PTDM more rapidly (76). Hypomagnesemia was associated with PTDM independent of the immunosuppressant regimen used. In this study, tacrolimus was associated with PTDM, but the association disappeared when controlling for hypomagnesemia, suggesting that tacrolimus may increase the risk of PTDM, in part through the induction of hypomagnesemia. A smaller retrospective study revealed similar findings (77). A study in liver transplant recipients also suggested that early posttransplant hypomagnesemia was associated with increased PTDM (78). A small clinical trial of magnesium supplementation was conducted to determine the relative impact of hypomagnesemia on PTDM in CNI-treated kidney transplant recipients. Unfortunately, the results were inconclusive. Magnesium supplementation resulted in a lower fasting glucose without a change in insulin resistance, which might suggest that hypomagnesemia does contribute. However, because there was a significant difference in fasting glucose between the two groups at baseline, the results were not definitive and further studies are still needed (79). In summary, CNIs have been associated with the risk of PTDM in kidney transplant recipients as a whole and in animal studies. Studies performed predominantly in vitro and in animal models suggest that CNIs may increase the risk of PTDM by reducing insulin secretion, increasing insulin resistance through both insulin signaling and noninsulin signaling mechanisms, or indirectly through its impact on magnesium concentration. However, small clinical studies of thin, euglycemic subjects did not demonstrate a significant impact of CNIs on insulin action, suggesting that pre-existing risk or susceptibility may be required. Sirolimus, the first mtor inhibitor to be used in solid organ transplantation, has also been associated with glucose intolerance after organ transplant. Soon after its introduction, patients treated with sirolimus were observed to have severe hypertriglyceridemia similar to that observed with other agents known to aggravate insulin resistance, raising suspicion that it could affect insulin action (80). Studies of data from the U.S. Renal Database suggest that sirolimus is independently associated with increased risk for diabetes after kidney transplant (25, 81, 82). A 10-year retrospective analysis of immunosuppression regimens also revealed that regimens that included sirolimus were more likely to develop diabetes after kidney transplantation (83). However, the impact of sirolimus may depend on individual or pre-existing susceptibility or may overall be less than that of the CNIs because case reports suggest that conversion from cyclosporine to sirolimus can improve glycemic control (84). Several mechanisms have been proposed. Sirolimus treatment has been shown to reduce -cell mass of human and rat islets through apoptosis (85). Sirolimus also has antiproliferative effects on multiple cells. In a pregnant mouse model, sirolimus impaired the proliferation of -cells, so it may also impact -cell replication in nonpregnant adults (86). Sirolimus has also been shown to impair proliferation of pancreatic ductal cells, which some believe serve as a precursor for the development of new islets (87).

8 44 Shivaswamy et al Post-Transplant Diabetes Mellitus Endocrine Reviews, February 2016, 37(1):37 61 However, the greatest impact of sirolimus on glucose metabolism may be on insulin signal transduction (88, 89) (Figure 3). The insulin receptor is composed of two extracellular -subunits, containing insulin binding sites, and two intracellular -subunits with tyrosine kinase activity. Insulin receptor binding triggers insulin receptor tyrosine autophosphorylation. This triggers phosphorylation of the docking protein family, known as the IRS proteins, which activates the phosphatidylinositol 3 kinase (PI3K) pathway and subsequent translocation of PI3K-dependent effector enzymes, such as phosphoinositide-dependent protein kinase 1 and Akt. After phosphorylation of Akt (at serine 473 and threonine 308), its activation is key to promotion of protein synthesis. This requires stimulation of the mtor-containing complex (mtorc1) and its substrate p70 ribosomal protein S6 kinase. Sirolimus, also called rapamycin, binds to mtor, and one of the important negative feedback mechanisms of this pathway is via protein kinase-dependent phosphorylation of IRS-1 at specific serine residues (307, 612, and 636, among others), leading to suppression of PI3K/Akt signaling. mtor exists in two multimeric complexes, mtorc1 and mtorc2 (90 92). mtorc1 is comprised of mtor, raptor, mlst8/g L, and PRAS40 (proline-rich Akt substrate 40). It regulates the phosphorylation of p70 ribosomal protein S6 kinase and promotes protein synthesis. mtorc2 consists of mtor, mlst8/g L, rictor, PROTOR, and msin1 and it plays a role in phosphorylation of Akt at serine 473, an event required for optimal Akt activity. In normal Sprague-Dawley rats, we showed that sirolimus treatment resulted in a dose-dependent increase in hyperglycemia accompanied by relative hyperinsulinemia and a dose-dependent increase in insulin secretion in response to an oral glucose challenge, both suggesting insulin resistance (56). Sirolimus suppresses insulin-stimulated Akt phosphorylation in the liver, fat, and muscle of male and female rats (54, 55). Sirolimus also disrupts the phosphorylation of mtorc2, reducing insulin-mediated suppression of hepatic gluconeogenesis (93), and can induce Yin Yang 1 dephosphorylation, which mediates suppression of insulin signaling genes, resulting in decreased insulin signaling (94). Finally, sirolimus has been shown in vitro to reduce -cell function and islet mass (37, 55, 85, 86). Although low-dose sirolimus can inhibit mtor1, responsible for its immunosuppressive effects, high-dose sirolimus may be required to inhibit mtor2, which is linked to insulin resistance (93, 95). However, not all studies have reported an impact of sirolimus on insulin action or glucose metabolism. In a study of normal, thin, human controls given a single dose of oral sirolimus, insulin-mediated glucose uptake was increased under somatostatininsulin clamp conditions used to simulate a postprandial environment, but not at baseline, and there was no impact on glucose production in either fasting or somatostatin clamp conditions (96). In pancreatectomized dogs made euglycemic with intrasplenic autoislet transplant, daily oral sirolimus treatment for 30 days (1 mg/kg/d) resulted in fasting and stimulated hyperinsulinemia although glucose uptake was increased and glucose concentration was normal, which was interpreted by the authors to suggest no significant impact on islet function. In this same model, daily im cyclosporine treatment for 30 days (12 mg/kg/d) also had no impact on glucose homeostasis. After allo-islet transplant of nonhuman primates treated with calcineurin-free, sirolimus immunosuppression, insulin secretion was reduced, as was glucagon response to arginine, although the impact of reduced islet mass cannot easily be discriminated from that of sirolimus treatment in this setting. Notably, at 2 to 3 weeks after transplant, insulin sensitivity was unchanged in these animals compared to their naive state before streptozotocin treatment (97, 98). Finally, studies 6 to 7 months after human islet transplant treated with low-dose tacrolimus and sirolimus immunosuppression also suggested normal insulin sensitivity at the liver and skeletal muscle (99). It should be noted that these negative studies have been performed in animal models and human subjects with presumed normal insulin sensitivity, whether normal controls or islet transplant recipients, who are often thinner than most type 1 diabetes patients. The absence of an effect in these studies again raises the question of whether underlying susceptibility to type 2 diabetes risk is required, as would be more likely to be observed in the kidney transplant and many other organ transplant populations. Time on medication may also be a factor because studies done acutely have not shown an impact. Some studies have already alluded to a potential biphasic effect of time, where an increase in insulin sensitivity was observed acutely after sirolimus treatment in C2C12 myotubes (100), L6 skeletal muscle cells (101), and healthy humans (96), possibly mediated by disrupting an S6-mediated feedback loop (100), whereas chronic administration induces insulin resistance in vitro (in C2C12 myotubes) (100) and in vivo (56, 93, 102), most likely due to disruption of mtorc2 (93). Everolimus is another available mtor inhibitor. Although there are fewer studies evaluating its relative impact on glucose metabolism, its mechanism of action, there are no data to suggest that it has any substantially different impact on future diabetes risk than sirolimus. D. Potential role of stress, inflammation, and infection Acute and chronic systemic inflammation have long been shown to be risk factors for the development of type

9 doi: /er press.endocrine.org/journal/edrv 45 2 diabetes (103). Solid organ transplantation is, by definition, an inflammatory state because of ongoing grafthost response due to acute and/or chronic rejection, reduced renal function common in this setting, and great incidence of and/or chronic infections associated with immunosuppression. Some of these potential causes of inflammation that may contribute to glucose intolerance and the development of PTDM are summarized below. Acute and chronic rejection is associated with risk of PTDM after kidney transplant (104, 105). However, cause and effect are harder to prove because rejection is also generally treated with high-dose corticosteroids, which can also contribute to risk (18, 104, 106, 107). In kidney transplant recipients, the source of the donor (deceased vs living) is a factor for PTDM. Deceased donor kidney allografts express higher levels of proinflammatory cytokines compared to living kidney donor grafts, whether related or unrelated, suggesting the graft injury that is more likely with a deceased donor is more important than relative histocompatibility to rejection. This proinflammatory response may contribute to the observation that kidney transplant recipients who receive a deceased donor graft rather than a living donor graft are also at greater risk for PTDM ( ). In a retrospective analysis of 386 kidney transplant recipients, those who received deceased donor grafts were nearly four times more likely to develop PTDM than recipients of living donor grafts (15), as confirmed in other studies (112, 113). Although one commonly cited study did not find an association between graft type and PTDM, it was published before the International Consensus Guidelines were established for diagnosis of PTDM, and the only criterion described for PTDM was initiation of hypoglycemic agent(s) (14). Infections are common after transplantation and are another source of inflammation. Two infections in particular have been observed to contribute to PTDM risk. One is hepatitis C virus (HCV), well established to be associated with the risk of diabetes in nontransplant patients (114). HCV infection is a common cause of chronic liver disease, the most common cause of cirrhosis requiring liver transplantation (115). Those receiving liver transplant for HCV-associated cirrhosis are known to have persistent HCV infection after transplant (116), and HCV has been consistently associated with an increased relative risk of PTDM (2.5- to 4-fold) after liver transplant ( ). HCV infection after liver transplant is associated with increased insulin resistance, as measured by homeostasis model assessment of insulin resistance, even when controlling for BMI, medications, alcohol, or degree of liver fibrosis (120). The burden of infection is a factor because insulin resistance is reported to develop sooner in recipients with the highest HCV RNA levels (120). HCV is also a risk factor for PTDM after kidney transplant. In a meta-analysis of 10 studies, Fabrizi et al (121) reported an increased risk of PTDM in kidney transplant recipients with HCV infection compared to HCV-negative recipients (odds ratio, 3.97; 95% confidence interval, ). HCV was also associated with risk after controlling for known risk factors in another cohort of 306 kidney transplant recipients (122) and a prospective observational trial (odds ratio, 6.37; 95% confidence interval, ) (123). As in liver transplant recipients, HCV-positive kidney recipients had reduced insulin sensitivity by minimal model estimate compared to matched HCV-negative patients (124). Further support for HCV as a risk factor for PTDM comes from small studies suggesting that treatment to clear HCV before kidney transplant may reduce the risk of PTDM after transplant (125, 126). In liver and kidney transplant recipients, the risk of PTDM is even higher in those who have HCV and are treated with tacrolimus-based immunosuppression, compared to either risk factor alone (40, 127, 128). Cytomegalovirus (CMV) is another infection associated with PTDM. CMV infection is more common after transplant due to immunosuppression. In one retrospective study of kidney transplant recipients who underwent OGTT at 10 weeks after transplant, those known to be CMV-positive demonstrated greater PTDM than those who were not (129). However, other studies did not show that CMV increased the risk of PTDM (9, 130, 131). These differences may be explained by differences in the size of studies, immunosuppressant regimens used that may impact PTDM risk, and methods used to diagnosis PTDM. However, a meta-analysis of seven kidney transplant studies incorporating 1389 recipients suggested that overall, CMV-positive recipients were at greater risk for PTDM (132). In a prospective observational trial of recipients with or without CMV, CMV infection was an independent risk factor for PTDM, whether or not the recipients were symptomatic, as assessed by OGTT at 10 weeks after transplant (133). First-phase, second-phase, and area-under-the-curve insulin secretion were all lower in CMVpositive recipients (133). CMV infection has also been associated with increased risk for PTDM after liver transplant, although in few studies (78, 118). To date, CMV infection has not yet been suggested to be a significant contributing factor for PTDM after heart or lung transplant. Although several potential mechanisms for these CMV effects include CMV-induced or leukocyte-mediated damage or destruction of -cells as well as proinflammatory cytokine production, none have been studied intensively (134).

10 46 Shivaswamy et al Post-Transplant Diabetes Mellitus Endocrine Reviews, February 2016, 37(1):37 61 E. Potential role of vitamin D or other factors Vitamin D deficiency has been associated with increased risk of diabetes in the nontransplant population (135, 136). A randomized controlled trial is currently investigating vitamin D replacement as a method to prevent progression of prediabetes to diabetes (Vitamin D and type 2 diabetes study [D2d] Identifier NCT ). However, whether vitamin D deficiency is a risk factor for PTDM has not been established. A randomized controlled trial of vitamin D supplementation in kidney transplant recipients is ongoing in France, with development of PTDM being one of the primary end points of the study (137). Results from this study will hopefully shed light on the potential association between vitamin D deficiency and PTDM risk. Statins are commonly used for treatment of hypercholesterolemia after transplant. Statins have also recently been shown to increase the incidence of new-onset diabetes outside of transplant groups. Statins have been associated with increased risk of impaired fasting glucose (IFG) and dysglycemia (IFG and/or PTDM) in renal allograft recipients (138). Patients treated with atorvastatin had more IFG and PTDM than those on fluvastatin (138). Statins have also been shown to increase the risk of PTDM in liver transplant recipients, especially in those with IFG before transplant (139). There are fewer studies of PTDM risk outside kidney transplantation. Because there are more systematic studies of transplant patients, particularly in nonkidney transplant populations, other factors may still be identified, some of which may be specific to those groups. IV. The Impact of PTDM on Transplant Outcomes A. Introduction Because diabetes and glucose intolerance alone have both been associated with adverse long-term outcomes after transplant in many groups, there is concern that PTDM may also impact outcomes after transplantation. These studies will necessarily depend, in part, on how and when PTDM is diagnosed. As noted previously, there was little consistency in the diagnosis of diabetes after transplant before the first International Consensus Guidelines were established in 2003, and there is still considerable variation in how centers screen for diabetes before and after transplant. Thus, many of the earliest studies of PTDM likely included patients who had unrecognized and previously untreated pretransplant diabetes (3). Because the diagnosis of PTDM was often transplant center-specific and often required a higher glucose threshold for a longer period of time than is used now, unrecognized or untreated hyperglycemia could itself impact outcomes. The impact of PTDM on transplant outcomes will likely also vary between types of organ transplant. Age and BMI of candidates and recipients differ between organ transplant groups, in addition to relative frequency of other cardiovascular risk factors and immunosuppression regimen, all of which impact PTDM risk. There is a much larger literature available regarding the impact of PTDM on transplant outcomes after kidney than other transplant groups. Fewer reports are likely more a reflection of inadequate long-term data of patients with PTDM to assess outcomes than a lack of impact. However, with greater recognition of PTDM, earlier diagnosis, and improved treatment options and methods, the relative impact of PTDM on transplant outcomes may also improve over time. We have summarized available published studies by transplant group below. B. Kidney transplant outcomes The first report to suggest that PTDM impacts survival showed that 1-year survival was 98% in those without PTDM vs 83% in those with PTDM (140). Age was also a factor in a retrospective study of 939 kidney transplant recipients wherein PTDM increased mortality in those less than 55 years of age, but not in those over 55, compared to non-ptdm (141). Awareness of the potential impact of post-transplant diabetes increased even more with the study by Cosio et al (142), who described a 2-fold increase in mortality (Figure 4) compared to nontransplant recipients, which was equal to that of pretransplant diabetes, already established to worsen outcome, and independent of other factors known to reduce survival, and was confirmed by Kaskiske et al and others (33, 34, 142, 143). The reduction in patient survival due to PTDM is largely attributable to increased cardiovascular disease events (33), as much as 3-fold even when controlling for other risk factors ( ). Most, but not all, studies also suggest that PTDM is associated with reduced graft survival, including a study of the United Renal Data System database (141, 147, 148). Although small studies suggest that PTDM is associated with higher acute rejection episode rates after kidney transplant (105, 149), it is difficult to determine whether PTDM causes rejection or whether the treatment of graft rejection, often requiring corticosteroids, results in greater risk for PTDM. However, the greatest impact of PTDM on graft survival is likely not due to increased rejection rates, but rather to death with a functioning graft (150). Only one study suggests PTDM to be a risk factor for deathcensored renal graft failure (143). Diabetes can predispose to many types of infection, as can immunosuppression. Thus, it is not surprising that

11 doi: /er press.endocrine.org/journal/edrv 47 Figure 4. Figure 4. Impact of PTDM on kidney transplant survival. Kaplan-Meier patient survival in the three study groups: no diabetes (thin line), pretransplant diabetes (thick line), and PTDM (dashed line). A, Survival was calculated from the day of transplantation in all three groups of patients. B, For PTDM, survival was calculated from the time of development of diabetes, and for the other two groups, from the day of transplantation. [Reprinted from F. G. Cosio et al: Patient survival after renal transplantation: IV. Impact of post-transplant diabetes. Kidney Int. 2002;62: (142), with permission. International Society of Nephrology.] PTDM has been shown to be associated with greater risk of sepsis and sepsis-related mortality (113, 148). PTDM is also a risk factor for developing CMV infection (15, 105). There are inadequate data on whether PTDM increases long-term risk of diabetic microvascular complications. However, a large retrospective study of kidney transplant recipients with PTDM found microvascular complications to be common, so diabetic microvascular complications may develop more quickly with PTDM than nontransplant diabetes, or that many with PTDM had unrecognized, pre-existing diabetes for years prior to transplant (151). C. Outcomes after other organ transplant groups To date, there are few studies of outcomes after heart transplant in those with or without PTDM, and most have been retrospective. In one single-center retrospective study of heart transplant patients in Spain, PTDM was associated with a higher prevalence of hypertension (36 vs 23%; P.001) and rejection (70 vs 45%; P.001) compared to nondiabetics (152). In another singlecenter retrospective study of heart transplant patients, PTDM was associated with a greater incidence of post-transplant renal failure (P.001) (153). There were no differences in overall survival in either study. PTDM has also been noted to increase the incidence of infection, but not survival, after heart transplantation in another study (154). In a Korean single-center study of heart transplants, the 5-year incidence of late infection (after 6 mo) was also higher in those with PTDM than those without (P.031) (154), but there was no difference in the 5-year overall survival rate. In summary, in the few available retrospective studies, PTDM after heart transplantation does not affect survival, but it may increase hypertension, renal failure, infection, and rejection. After liver transplant, a recent retrospective study of 438 recipients showed that PTDM was associated with increased risk of sepsis, chronic renal fail-