Changes Over Time in Hepatic Markers Predict Changes in Insulin Sensitivity, β-Cell Function, and Glycemia

Changes Over Time in Hepatic Markers Predict Changes in Insulin Sensitivity, β-Cell Function,... Abstract Context Serum concentrations of liver enzymes and the hepatokine fetuin-A have been linked to the risk of type 2 diabetes, but their longitudinal impact on insulin resistance and β-cell dysfunction is unclear. Objective To evaluate the impact of changes over 2 years in fetuin-A and the liver enzymes alanine aminotransferase (ALT), aspartate aminotransferase (AST), and γ-glutamyltransferase (GGT) on changes in insulin sensitivity, β-cell function, and glycemia in women with varying degrees of previous gestational dysglycemia, reflecting a range of future diabetic risk. Design/Setting/Participants In total, 336 women underwent glucose challenge test (GCT) and oral glucose tolerance test (OGTT) in pregnancy, followed by repeat OGTT and measurement of ALT/AST/GGT/fetuin-A at both 1 year and 3 years postpartum. The antepartum GCT/OGTT identified four gestational glucose tolerance groups: gestational diabetes (n = 104), gestational impaired glucose tolerance (n = 59), abnormal GCT with normal OGTT (n = 98), and normal GCT/OGTT (n = 75). Results At 1 and 3 years postpartum, ALT, AST, GGT, and fetuin-A did not differ across the four groups, but the intervening change in ALT/AST ratio was greater in the gestational dysglycemia groups (P = 0.05). Higher baseline ALT/AST (t = −1.99, P = 0.05) and fetuin-A (t = −3.17, P = 0.002) predicted lower insulin sensitivity (Matsuda) at 3 years, as did their respective changes from 1 to 3 years (ALT/AST: t = −5.47, P < 0.0001; fetuin-A: t = −3.56, P = 0.0004). Change in ALT/AST predicted lower β-cell function (t = −2.33, P = 0.02) and higher fasting glucose at 3 years (t = 2.55, P = 0.01). Moreover, baseline fetuin-A predicted prediabetes/diabetes at 3 years (OR, 1.38; 95% CI, 1.01 to 1.88). Conclusion Circulating hepatic markers, particularly ALT/AST ratio and fetuin-A, track with changes in insulin sensitivity and β-cell function, supporting a pathophysiologic basis in their prediction of diabetic risk. Over the past 15 years, several large prospective epidemiologic studies have shown that modest elevations of serum liver enzymes, such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), and γ-glutamyl transferase (GGT), are associated with an increased risk of subsequently developing type 2 diabetes (T2DM) (1–3). More recently, similar associations with incident T2DM have been reported with fetuin-A, a novel serum glycoprotein that is secreted by the liver into the circulation at high concentration (a “hepatokine”) (4–7). Collectively, it is believed that perturbations of these markers may be reflecting hepatic fat, the deposition of which is often enhanced in those who develop T2DM (8, 9). However, despite an abundance of epidemiologic studies in which baseline measurement of these hepatic markers has been associated with the subsequent development of T2DM, evidence supporting causality in this regard is limited. Notably, there is a relative paucity of human data linking changes in hepatic markers with changes in insulin sensitivity and β-cell function over time, as might be expected for causal mediators. Reasons for this evidence gap include (1) the fact that most studies have measured these markers at baseline only, (2) few have assessed insulin sensitivity and β-cell function, and (3) even fewer have evaluated changes over time in these factors. Thus, recognizing these limitations, we sought to evaluate the longitudinal relationship over time between hepatic markers and concurrent changes in insulin sensitivity and β-cell function in a population at risk for T2DM. Glucose intolerance in pregnancy provides the unique opportunity to identify just such a patient population. Indeed, each degree of antepartum glucose intolerance [ranging from gestational diabetes mellitus (GDM) to milder gestational impaired glucose tolerance (GIGT) to lesser dysglycemia to normoglycemia] identifies a proportionate level of future risk of T2DM (one that is highest in GDM, followed by GIGT, etc) (10–12). Moreover, each degree of gestational glucose intolerance predicts distinct trajectories of insulin sensitivity, β-cell function, and glycemia in the first 3 years postpartum (12). Accordingly, the early postpartum years following gestational dysglycemia can provide a model of the early natural history of T2DM with which to evaluate the impact of hepatic markers on glucose homeostasis. In this setting, we hypothesized that changes in hepatic makers would be associated with changes in insulin sensitivity and/or β-cell function. Thus, our objective in this study was to characterize the impact of changes between 1 and 3 years postpartum in liver enzymes (ALT, AST, GGT) and fetuin-A on changes in insulin sensitivity, β-cell function, and glycemia in women with varying degrees of previous gestational dysglycemia and hence a range of future diabetic risk. Methods The study population consisted of women participating in a prospective observational cohort study in which we are evaluating the relationship between glucose tolerance in pregnancy and metabolic function in the years after delivery. The study protocol has been described in detail (12). In brief, women are first recruited at the time of antepartum screening for GDM in the late second/early third trimester and undergo metabolic characterization at recruitment and again at 3 months and 1 year postpartum. At the latter visit, they are recruited into an ongoing long-term observational cohort study in which participating women undergo serial metabolic characterization biannually thereafter. This analysis reports on the associations of hepatic markers with metabolic function in 336 women who have completed their 3-year postpartum visit. The study protocol has been approved by the Mount Sinai Hospital Research Ethics Board, and all women have provided written informed consent for their participation. Recruitment and determination of glucose tolerance status in pregnancy At our institution, all pregnant women are screened for GDM by a 50-g glucose challenge test (GCT), followed by referral for a diagnostic oral glucose tolerance test (OGTT) if the GCT is abnormal (plasma glucose ≥7.8 mmol/L at 1 hour after ingestion of a 50-g glucose load). For this study, women are recruited either before or after the GCT, and all participants undergo a 3-hour 100-g OGTT for ascertainment of gestational glucose tolerance status, regardless of the GCT result (i.e., even if normal). As previously described (12), the recruitment of women after an abnormal GCT serves to enrich the study population for those with varying degrees of glucose intolerance. The GCT and OGTT enable stratification of participants into the following gestational glucose tolerance groups: (1) GDM, defined by National Diabetes Data Group (NDDG) criteria, which require at least two of the following on the OGTT: fasting blood glucose ≥5.8 mmol/L, 1-hour glucose ≥10.6 mmol/L, 2-hour glucose ≥9.2 mmol/L, or 3-hour glucose ≥8.1 mmol/L (2) GIGT, defined by meeting only one of the above NDDG criteria (3) Abnormal GCT with normal glucose tolerance (NGT), defined by an abnormal GCT followed by NGT on the OGTT (i.e., meeting none of the NDDG criteria) (4) Normal GCT NGT, defined by a normal GCT followed by NGT on the OGTT These four groups reflect the full spectrum of future diabetic risk and have been previously shown to predict distinct trajectories of insulin sensitivity, β-cell function, and glycemia in the first 3 years postpartum (12). Assessments at 1 and 3 years postpartum Participants return to the clinical investigation unit at 1 and 3 years for metabolic characterization, including 2-hour 75-g OGTT on both occasions (12). On each OGTT, current glucose tolerance status was defined according to Canadian Diabetes Association (CDA) guidelines (13). CDA guidelines define prediabetes as impaired glucose tolerance (IGT), impaired fasting glucose (IFG), or combined IFG/IGT (13). All OGTTs were performed in the morning after an overnight fast, with venous blood samples drawn for measurement of glucose and specific insulin at fasting and at 30, 60, and 120 minutes following ingestion of the glucose load. Specific insulin was measured with the Roche-Elecsys-1010 immunoassay analyzer and electrochemiluminescence immunoassay kit (Roche Diagnostics, Laval, QB, Canada). On each OGTT, the primary measure of insulin sensitivity was the Matsuda index (14), with the homeostasis model assessment of insulin resistance (HOMA-IR) providing a secondary measure (15). The primary measure of β-cell function was the insulinogenic index (IGI)/HOMA-IR (16), with the Insulin Secretion-Sensitivity Index–2 (ISSI-2) providing a secondary measure (17, 18). Fetuin-A was measured by an enzyme-linked immunosorbent assay (ALPCO, Salem, NH), with a lower limit of detection of 5.0 μg/L and an upper limit of detection of 216 μg/L. ALT, AST, and GGT were measured by the routine clinical biochemistry laboratory at Mount Sinai Hospital. Statistical analyses All analyses were conducted using SAS 9.2 (SAS Institute, Cary, NC). All tests were two-sided and performed at a significance level of P < 0.05. Characteristics of the gestational glucose tolerance groups were compared at 1 year and 3 years postpartum by either one-way analysis of variance or Kruskal-Wallis test for continuous variables, or either χ2 or Fisher exact test for categorical variables (Table 1). In particular, changes in hepatic markers from 1 to 3 years were compared between these groups (i.e., groups that reflect different degrees of diabetic risk). Table 1. Demographic, Clinical, and Metabolic Characteristics of Study Population at 1 Year and 3 Years Postpartum, Stratified Into the Following Four Groups Based on Gestational Glucose Tolerance Status: Normal GCT NGT, Abnormal GCT NGT, GIGT, and GDM Characteristic Normal GCT NGT (n = 75) Abnormal GCT NGT (n = 98) GIGT (n = 59) GDM (n = 104) P Value At 1 y postpartum  Age, y 36 ± 4 36 ± 4 36 ± 4 36 ± 4 0.82  Ethnicity, n (%) 0.50   White 57 (76.0) 73 (74.5) 40 (67.8) 69 (66.4)   Asian 5 (6.7) 9 (9.2) 7 (11.9) 17 (16.4)   Other 13 (17.3) 16 (16.3) 12 (20.3) 18 (17.3)  Family history of T2DM, n (%) 40 (53.3) 58 (59.2) 38 (64.4) 68 (65.4) 0.38  Months breastfeeding, mo 11 (6–12) 10 (6–12) 9 (4–12) 10 (3–12) 0.43  Current smoking, n (%) 2 (2.7) 3 (3.1) 5 (8.8) 2 (2.0) 0.21  BMI, kg/m2 24.4 (21.5–28.4) 23.7 (21.8–27.8) 25.4 (23.1–30.1) 25.5 (22.5–29.5) 0.12  Waist circumference, cm 87 ± 12 85 ± 12 89 ± 13 90 ± 14 0.04  Insulin sensitivity/resistance   Matsuda index 11.0 (6.4–15.5) 10.2 (6.1–13.8) 7.5 (4.9–12.9) 7.7 (4.4–11.0) 0.003   HOMA-IR 1.1 (0.7–1.7) 1.1 (0.6–1.7) 1.4 (0.8–2.0) 1.2 (0.7–2.0) 0.14  β-Cell function   ISSI-2 877 ± 292 841 ± 324 655 ± 227 673 ± 267 <0.0001   Insulinogenic index/HOMA-IR 13.7 (8.7–22.8) 10.6 (6.6–16.4) 7.0 (4.0–9.1) 7.3 (4.5–12.1) <0.0001  OGTT   Fasting glucose, mmol/L 4.6 ± 0.3 4.6 ± 0.4 4.9 ± 0.6 4.9 ± 0.5 <0.0001   2-h glucose, mmol/L 5.4 ± 1.2 6.1 ± 1.6 6.4 ± 1.8 6.9 ± 1.9 <0.0001  Current glucose tolerance, n (%) <0.0001   Normal 69 (97.2) 82 (87.2) 42 (77.8) 69 (70.4)   Prediabetes/diabetes 2 (2.8) 12 (12.8) 12 (22.2) 29 (29.6)  ALT, IU/L 11 (9–15) 11 (9–15) 12 (9–17) 12 (9–14) 0.81  AST, IU/L 18 (17–22) 19 (17–21) 19 (16–24) 19 (16–23) 0.97  ALT/AST ratio 0.71 ± 0.38 0.63 ± 0.21 0.65 ± 0.29 0.64 ± 0.28 0.41  GGT, IU/L 10 (8.0–15) 11 (8–14) 12 (8–17) 12 (8–17) 0.32  Fetuin-A, g/L 0.5 (0.4–0.5) 0.5 (0.4–0.6) 0.5 (0.4–0.6) 0.5 (0.4–0.6) 0.10 At 3 y postpartum  BMI, kg/m2 25.5 ± 4.5 25.4 ± 5.0 26.6 ± 4.8 26.9 ± 6.1 0.14  Waist circumference, cm 88 ± 12 86 ± 12 89 ± 12 90 ± 13 0.14  Insulin sensitivity/resistance   Matsuda index 10.0 (7.4–13.0) 8.2 (5.5–12.3) 7.3 (4.3–10.8) 6.2 (4.1–9.6) <0.0001   HOMA-IR 1.1 (0.7–1.8) 1.2 (0.7–1.9) 1.4 (1.0–2.1) 1.5 (1.0–2.4) 0.01  β-Cell function   ISSI-2 925 ± 331 898 ± 397 759 ± 333 668 ± 365 <0.0001   Insulinogenic index/HOMA-IR 13.1 (9.0–22.6) 10.7 (8.1–18.4) 8.7 (4.6–13.4) 6.8 (4.4–11.3) <0.0001  OGTT   Fasting glucose, mmol/L 4.6 ± 0.4 4.6 ± 0.5 4.8 ± 0.5 4.9 ± 0.6 <0.0001   2-h glucose, mmol/L 5.6 ± 1.2 6.1 ± 1.7 6.6 ± 2.1 7.4 ± 2.2 <0.0001  Current glucose tolerance, n (%) <0.0001   Normal 70 (93.3) 84 (85.7) 47 (80.0) 66 (63.5)   Prediabetes/diabetes 5 (6.7) 14 (14.3) 12 (20.0) 38 (36.5)  ALT, IU/L 15 (12–19) 15 (12–19) 15 (10–18) 16 (13–20) 0.69  AST, IU/L 18 (16–23) 18 (16–22) 17 (15–20) 18 (15–22) 0.34  ALT/AST ratio 0.86 ± 0.26 0.87 ± 0.28 0.90 ± 0.33 0.88 ± 0.22 0.80  GGT, IU/L 12 (9–17) 12 (10–17) 13 (10–18) 13 (11–22) 0.21  Fetuin-A, g/L 0.6 (0.5–0.7) 0.6 (0.5–0.8) 0.6 (0.5–0.8) 0.6 (0.5–0.7) 0.24 Changes from 1 to 3 y  Change in ALT 1.3 ± 11.7 4.4 ± 6.2 2.4 ± 10.7 3.8 ± 9.3 0.23  Change in AST −1.0 ± 8.6 −0.9 ± 5.5 −3.9 ± 8.1 −1.5 ± 7.5 0.11  Change in ALT/AST ratio 0.14 ± 0.36 0.26 ± 0.25 0.28 ± 0.32 0.25 ± 0.28 0.05  Change in GGT 0.8 ± 9.8 2.3 ± 7.1 4.6 ± 17.6 1.5 ± 12.6 0.38  Change in fetuin-A 0.1 ± 0.2 0.1 ± 0.3 0.1 ± 0.4 0.1 ± 0.3 0.98 Characteristic Normal GCT NGT (n = 75) Abnormal GCT NGT (n = 98) GIGT (n = 59) GDM (n = 104) P Value At 1 y postpartum  Age, y 36 ± 4 36 ± 4 36 ± 4 36 ± 4 0.82  Ethnicity, n (%) 0.50   White 57 (76.0) 73 (74.5) 40 (67.8) 69 (66.4)   Asian 5 (6.7) 9 (9.2) 7 (11.9) 17 (16.4)   Other 13 (17.3) 16 (16.3) 12 (20.3) 18 (17.3)  Family history of T2DM, n (%) 40 (53.3) 58 (59.2) 38 (64.4) 68 (65.4) 0.38  Months breastfeeding, mo 11 (6–12) 10 (6–12) 9 (4–12) 10 (3–12) 0.43  Current smoking, n (%) 2 (2.7) 3 (3.1) 5 (8.8) 2 (2.0) 0.21  BMI, kg/m2 24.4 (21.5–28.4) 23.7 (21.8–27.8) 25.4 (23.1–30.1) 25.5 (22.5–29.5) 0.12  Waist circumference, cm 87 ± 12 85 ± 12 89 ± 13 90 ± 14 0.04  Insulin sensitivity/resistance   Matsuda index 11.0 (6.4–15.5) 10.2 (6.1–13.8) 7.5 (4.9–12.9) 7.7 (4.4–11.0) 0.003   HOMA-IR 1.1 (0.7–1.7) 1.1 (0.6–1.7) 1.4 (0.8–2.0) 1.2 (0.7–2.0) 0.14  β-Cell function   ISSI-2 877 ± 292 841 ± 324 655 ± 227 673 ± 267 <0.0001   Insulinogenic index/HOMA-IR 13.7 (8.7–22.8) 10.6 (6.6–16.4) 7.0 (4.0–9.1) 7.3 (4.5–12.1) <0.0001  OGTT   Fasting glucose, mmol/L 4.6 ± 0.3 4.6 ± 0.4 4.9 ± 0.6 4.9 ± 0.5 <0.0001   2-h glucose, mmol/L 5.4 ± 1.2 6.1 ± 1.6 6.4 ± 1.8 6.9 ± 1.9 <0.0001  Current glucose tolerance, n (%) <0.0001   Normal 69 (97.2) 82 (87.2) 42 (77.8) 69 (70.4)   Prediabetes/diabetes 2 (2.8) 12 (12.8) 12 (22.2) 29 (29.6)  ALT, IU/L 11 (9–15) 11 (9–15) 12 (9–17) 12 (9–14) 0.81  AST, IU/L 18 (17–22) 19 (17–21) 19 (16–24) 19 (16–23) 0.97  ALT/AST ratio 0.71 ± 0.38 0.63 ± 0.21 0.65 ± 0.29 0.64 ± 0.28 0.41  GGT, IU/L 10 (8.0–15) 11 (8–14) 12 (8–17) 12 (8–17) 0.32  Fetuin-A, g/L 0.5 (0.4–0.5) 0.5 (0.4–0.6) 0.5 (0.4–0.6) 0.5 (0.4–0.6) 0.10 At 3 y postpartum  BMI, kg/m2 25.5 ± 4.5 25.4 ± 5.0 26.6 ± 4.8 26.9 ± 6.1 0.14  Waist circumference, cm 88 ± 12 86 ± 12 89 ± 12 90 ± 13 0.14  Insulin sensitivity/resistance   Matsuda index 10.0 (7.4–13.0) 8.2 (5.5–12.3) 7.3 (4.3–10.8) 6.2 (4.1–9.6) <0.0001   HOMA-IR 1.1 (0.7–1.8) 1.2 (0.7–1.9) 1.4 (1.0–2.1) 1.5 (1.0–2.4) 0.01  β-Cell function   ISSI-2 925 ± 331 898 ± 397 759 ± 333 668 ± 365 <0.0001   Insulinogenic index/HOMA-IR 13.1 (9.0–22.6) 10.7 (8.1–18.4) 8.7 (4.6–13.4) 6.8 (4.4–11.3) <0.0001  OGTT   Fasting glucose, mmol/L 4.6 ± 0.4 4.6 ± 0.5 4.8 ± 0.5 4.9 ± 0.6 <0.0001   2-h glucose, mmol/L 5.6 ± 1.2 6.1 ± 1.7 6.6 ± 2.1 7.4 ± 2.2 <0.0001  Current glucose tolerance, n (%) <0.0001   Normal 70 (93.3) 84 (85.7) 47 (80.0) 66 (63.5)   Prediabetes/diabetes 5 (6.7) 14 (14.3) 12 (20.0) 38 (36.5)  ALT, IU/L 15 (12–19) 15 (12–19) 15 (10–18) 16 (13–20) 0.69  AST, IU/L 18 (16–23) 18 (16–22) 17 (15–20) 18 (15–22) 0.34  ALT/AST ratio 0.86 ± 0.26 0.87 ± 0.28 0.90 ± 0.33 0.88 ± 0.22 0.80  GGT, IU/L 12 (9–17) 12 (10–17) 13 (10–18) 13 (11–22) 0.21  Fetuin-A, g/L 0.6 (0.5–0.7) 0.6 (0.5–0.8) 0.6 (0.5–0.8) 0.6 (0.5–0.7) 0.24 Changes from 1 to 3 y  Change in ALT 1.3 ± 11.7 4.4 ± 6.2 2.4 ± 10.7 3.8 ± 9.3 0.23  Change in AST −1.0 ± 8.6 −0.9 ± 5.5 −3.9 ± 8.1 −1.5 ± 7.5 0.11  Change in ALT/AST ratio 0.14 ± 0.36 0.26 ± 0.25 0.28 ± 0.32 0.25 ± 0.28 0.05  Change in GGT 0.8 ± 9.8 2.3 ± 7.1 4.6 ± 17.6 1.5 ± 12.6 0.38  Change in fetuin-A 0.1 ± 0.2 0.1 ± 0.3 0.1 ± 0.4 0.1 ± 0.3 0.98 P values are for overall comparison across groups by one-way analysis of variance or Kruskal-Wallis test for continuous variables, or either χ2 or Fisher exact test for categorical variables. Continuous variables are presented as mean ± SD (if normally distributed) or median with interquartile range (if skewed). View Large Table 1. Demographic, Clinical, and Metabolic Characteristics of Study Population at 1 Year and 3 Years Postpartum, Stratified Into the Following Four Groups Based on Gestational Glucose Tolerance Status: Normal GCT NGT, Abnormal GCT NGT, GIGT, and GDM Characteristic Normal GCT NGT (n = 75) Abnormal GCT NGT (n = 98) GIGT (n = 59) GDM (n = 104) P Value At 1 y postpartum  Age, y 36 ± 4 36 ± 4 36 ± 4 36 ± 4 0.82  Ethnicity, n (%) 0.50   White 57 (76.0) 73 (74.5) 40 (67.8) 69 (66.4)   Asian 5 (6.7) 9 (9.2) 7 (11.9) 17 (16.4)   Other 13 (17.3) 16 (16.3) 12 (20.3) 18 (17.3)  Family history of T2DM, n (%) 40 (53.3) 58 (59.2) 38 (64.4) 68 (65.4) 0.38  Months breastfeeding, mo 11 (6–12) 10 (6–12) 9 (4–12) 10 (3–12) 0.43  Current smoking, n (%) 2 (2.7) 3 (3.1) 5 (8.8) 2 (2.0) 0.21  BMI, kg/m2 24.4 (21.5–28.4) 23.7 (21.8–27.8) 25.4 (23.1–30.1) 25.5 (22.5–29.5) 0.12  Waist circumference, cm 87 ± 12 85 ± 12 89 ± 13 90 ± 14 0.04  Insulin sensitivity/resistance   Matsuda index 11.0 (6.4–15.5) 10.2 (6.1–13.8) 7.5 (4.9–12.9) 7.7 (4.4–11.0) 0.003   HOMA-IR 1.1 (0.7–1.7) 1.1 (0.6–1.7) 1.4 (0.8–2.0) 1.2 (0.7–2.0) 0.14  β-Cell function   ISSI-2 877 ± 292 841 ± 324 655 ± 227 673 ± 267 <0.0001   Insulinogenic index/HOMA-IR 13.7 (8.7–22.8) 10.6 (6.6–16.4) 7.0 (4.0–9.1) 7.3 (4.5–12.1) <0.0001  OGTT   Fasting glucose, mmol/L 4.6 ± 0.3 4.6 ± 0.4 4.9 ± 0.6 4.9 ± 0.5 <0.0001   2-h glucose, mmol/L 5.4 ± 1.2 6.1 ± 1.6 6.4 ± 1.8 6.9 ± 1.9 <0.0001  Current glucose tolerance, n (%) <0.0001   Normal 69 (97.2) 82 (87.2) 42 (77.8) 69 (70.4)   Prediabetes/diabetes 2 (2.8) 12 (12.8) 12 (22.2) 29 (29.6)  ALT, IU/L 11 (9–15) 11 (9–15) 12 (9–17) 12 (9–14) 0.81  AST, IU/L 18 (17–22) 19 (17–21) 19 (16–24) 19 (16–23) 0.97  ALT/AST ratio 0.71 ± 0.38 0.63 ± 0.21 0.65 ± 0.29 0.64 ± 0.28 0.41  GGT, IU/L 10 (8.0–15) 11 (8–14) 12 (8–17) 12 (8–17) 0.32  Fetuin-A, g/L 0.5 (0.4–0.5) 0.5 (0.4–0.6) 0.5 (0.4–0.6) 0.5 (0.4–0.6) 0.10 At 3 y postpartum  BMI, kg/m2 25.5 ± 4.5 25.4 ± 5.0 26.6 ± 4.8 26.9 ± 6.1 0.14  Waist circumference, cm 88 ± 12 86 ± 12 89 ± 12 90 ± 13 0.14  Insulin sensitivity/resistance   Matsuda index 10.0 (7.4–13.0) 8.2 (5.5–12.3) 7.3 (4.3–10.8) 6.2 (4.1–9.6) <0.0001   HOMA-IR 1.1 (0.7–1.8) 1.2 (0.7–1.9) 1.4 (1.0–2.1) 1.5 (1.0–2.4) 0.01  β-Cell function   ISSI-2 925 ± 331 898 ± 397 759 ± 333 668 ± 365 <0.0001   Insulinogenic index/HOMA-IR 13.1 (9.0–22.6) 10.7 (8.1–18.4) 8.7 (4.6–13.4) 6.8 (4.4–11.3) <0.0001  OGTT   Fasting glucose, mmol/L 4.6 ± 0.4 4.6 ± 0.5 4.8 ± 0.5 4.9 ± 0.6 <0.0001   2-h glucose, mmol/L 5.6 ± 1.2 6.1 ± 1.7 6.6 ± 2.1 7.4 ± 2.2 <0.0001  Current glucose tolerance, n (%) <0.0001   Normal 70 (93.3) 84 (85.7) 47 (80.0) 66 (63.5)   Prediabetes/diabetes 5 (6.7) 14 (14.3) 12 (20.0) 38 (36.5)  ALT, IU/L 15 (12–19) 15 (12–19) 15 (10–18) 16 (13–20) 0.69  AST, IU/L 18 (16–23) 18 (16–22) 17 (15–20) 18 (15–22) 0.34  ALT/AST ratio 0.86 ± 0.26 0.87 ± 0.28 0.90 ± 0.33 0.88 ± 0.22 0.80  GGT, IU/L 12 (9–17) 12 (10–17) 13 (10–18) 13 (11–22) 0.21  Fetuin-A, g/L 0.6 (0.5–0.7) 0.6 (0.5–0.8) 0.6 (0.5–0.8) 0.6 (0.5–0.7) 0.24 Changes from 1 to 3 y  Change in ALT 1.3 ± 11.7 4.4 ± 6.2 2.4 ± 10.7 3.8 ± 9.3 0.23  Change in AST −1.0 ± 8.6 −0.9 ± 5.5 −3.9 ± 8.1 −1.5 ± 7.5 0.11  Change in ALT/AST ratio 0.14 ± 0.36 0.26 ± 0.25 0.28 ± 0.32 0.25 ± 0.28 0.05  Change in GGT 0.8 ± 9.8 2.3 ± 7.1 4.6 ± 17.6 1.5 ± 12.6 0.38  Change in fetuin-A 0.1 ± 0.2 0.1 ± 0.3 0.1 ± 0.4 0.1 ± 0.3 0.98 Characteristic Normal GCT NGT (n = 75) Abnormal GCT NGT (n = 98) GIGT (n = 59) GDM (n = 104) P Value At 1 y postpartum  Age, y 36 ± 4 36 ± 4 36 ± 4 36 ± 4 0.82  Ethnicity, n (%) 0.50   White 57 (76.0) 73 (74.5) 40 (67.8) 69 (66.4)   Asian 5 (6.7) 9 (9.2) 7 (11.9) 17 (16.4)   Other 13 (17.3) 16 (16.3) 12 (20.3) 18 (17.3)  Family history of T2DM, n (%) 40 (53.3) 58 (59.2) 38 (64.4) 68 (65.4) 0.38  Months breastfeeding, mo 11 (6–12) 10 (6–12) 9 (4–12) 10 (3–12) 0.43  Current smoking, n (%) 2 (2.7) 3 (3.1) 5 (8.8) 2 (2.0) 0.21  BMI, kg/m2 24.4 (21.5–28.4) 23.7 (21.8–27.8) 25.4 (23.1–30.1) 25.5 (22.5–29.5) 0.12  Waist circumference, cm 87 ± 12 85 ± 12 89 ± 13 90 ± 14 0.04  Insulin sensitivity/resistance   Matsuda index 11.0 (6.4–15.5) 10.2 (6.1–13.8) 7.5 (4.9–12.9) 7.7 (4.4–11.0) 0.003   HOMA-IR 1.1 (0.7–1.7) 1.1 (0.6–1.7) 1.4 (0.8–2.0) 1.2 (0.7–2.0) 0.14  β-Cell function   ISSI-2 877 ± 292 841 ± 324 655 ± 227 673 ± 267 <0.0001   Insulinogenic index/HOMA-IR 13.7 (8.7–22.8) 10.6 (6.6–16.4) 7.0 (4.0–9.1) 7.3 (4.5–12.1) <0.0001  OGTT   Fasting glucose, mmol/L 4.6 ± 0.3 4.6 ± 0.4 4.9 ± 0.6 4.9 ± 0.5 <0.0001   2-h glucose, mmol/L 5.4 ± 1.2 6.1 ± 1.6 6.4 ± 1.8 6.9 ± 1.9 <0.0001  Current glucose tolerance, n (%) <0.0001   Normal 69 (97.2) 82 (87.2) 42 (77.8) 69 (70.4)   Prediabetes/diabetes 2 (2.8) 12 (12.8) 12 (22.2) 29 (29.6)  ALT, IU/L 11 (9–15) 11 (9–15) 12 (9–17) 12 (9–14) 0.81  AST, IU/L 18 (17–22) 19 (17–21) 19 (16–24) 19 (16–23) 0.97  ALT/AST ratio 0.71 ± 0.38 0.63 ± 0.21 0.65 ± 0.29 0.64 ± 0.28 0.41  GGT, IU/L 10 (8.0–15) 11 (8–14) 12 (8–17) 12 (8–17) 0.32  Fetuin-A, g/L 0.5 (0.4–0.5) 0.5 (0.4–0.6) 0.5 (0.4–0.6) 0.5 (0.4–0.6) 0.10 At 3 y postpartum  BMI, kg/m2 25.5 ± 4.5 25.4 ± 5.0 26.6 ± 4.8 26.9 ± 6.1 0.14  Waist circumference, cm 88 ± 12 86 ± 12 89 ± 12 90 ± 13 0.14  Insulin sensitivity/resistance   Matsuda index 10.0 (7.4–13.0) 8.2 (5.5–12.3) 7.3 (4.3–10.8) 6.2 (4.1–9.6) <0.0001   HOMA-IR 1.1 (0.7–1.8) 1.2 (0.7–1.9) 1.4 (1.0–2.1) 1.5 (1.0–2.4) 0.01  β-Cell function   ISSI-2 925 ± 331 898 ± 397 759 ± 333 668 ± 365 <0.0001   Insulinogenic index/HOMA-IR 13.1 (9.0–22.6) 10.7 (8.1–18.4) 8.7 (4.6–13.4) 6.8 (4.4–11.3) <0.0001  OGTT   Fasting glucose, mmol/L 4.6 ± 0.4 4.6 ± 0.5 4.8 ± 0.5 4.9 ± 0.6 <0.0001   2-h glucose, mmol/L 5.6 ± 1.2 6.1 ± 1.7 6.6 ± 2.1 7.4 ± 2.2 <0.0001  Current glucose tolerance, n (%) <0.0001   Normal 70 (93.3) 84 (85.7) 47 (80.0) 66 (63.5)   Prediabetes/diabetes 5 (6.7) 14 (14.3) 12 (20.0) 38 (36.5)  ALT, IU/L 15 (12–19) 15 (12–19) 15 (10–18) 16 (13–20) 0.69  AST, IU/L 18 (16–23) 18 (16–22) 17 (15–20) 18 (15–22) 0.34  ALT/AST ratio 0.86 ± 0.26 0.87 ± 0.28 0.90 ± 0.33 0.88 ± 0.22 0.80  GGT, IU/L 12 (9–17) 12 (10–17) 13 (10–18) 13 (11–22) 0.21  Fetuin-A, g/L 0.6 (0.5–0.7) 0.6 (0.5–0.8) 0.6 (0.5–0.8) 0.6 (0.5–0.7) 0.24 Changes from 1 to 3 y  Change in ALT 1.3 ± 11.7 4.4 ± 6.2 2.4 ± 10.7 3.8 ± 9.3 0.23  Change in AST −1.0 ± 8.6 −0.9 ± 5.5 −3.9 ± 8.1 −1.5 ± 7.5 0.11  Change in ALT/AST ratio 0.14 ± 0.36 0.26 ± 0.25 0.28 ± 0.32 0.25 ± 0.28 0.05  Change in GGT 0.8 ± 9.8 2.3 ± 7.1 4.6 ± 17.6 1.5 ± 12.6 0.38  Change in fetuin-A 0.1 ± 0.2 0.1 ± 0.3 0.1 ± 0.4 0.1 ± 0.3 0.98 P values are for overall comparison across groups by one-way analysis of variance or Kruskal-Wallis test for continuous variables, or either χ2 or Fisher exact test for categorical variables. Continuous variables are presented as mean ± SD (if normally distributed) or median with interquartile range (if skewed). View Large Next, in Table 2, Spearman partial correlation analyses were conducted to assess the relationships between baseline-adjusted changes in each of the hepatic markers from 1 to 3 years with concurrent baseline-adjusted changes in metabolic factors (anthropometrics, insulin sensitivity/resistance, β-cell function, glycemia). We then proceeded to multiple linear regression analyses to determine whether changes in hepatic markers from 1 to 3 years were independently associated with the Matsuda index at 3 years (Table 3) and IGI/HOMA-IR at 3 years (Table 4) as per our main hypothesis, after complete adjustment for covariates. For each of these outcomes, we constructed the following five models to test the hepatic markers in turn: ALT (model I), AST (model II), ALT/AST ratio (model III), GGT (model IV), and fetuin-A (model V). Each regression model was constructed with the following covariates: (1) clinical risk factors for diabetes [age, ethnicity, family history of diabetes, body mass index (BMI) at 1 year, change in BMI from 1 to 3 years, and duration of breastfeeding], (2) the baseline measure of the outcome variable at 1 year, and (3) the indicated hepatic marker at 1 year and its change from 1 to 3 years. Multiple linear regression analyses were similarly conducted for the secondary measures of insulin sensitivity (HOMA-IR) and β-cell function (ISSI-2) in Supplemental Tables 1 and 2, respectively. We then performed multiple linear regression analyses to determine whether changes in hepatic markers from 1 to 3 years were independently associated with fasting glucose at 3 years (Table 5), with model construction performed in the same way. Table 2. Partial Spearman Correlations of Baseline-Adjusted Changes in Hepatic Markers (ALT, AST, ALT/AST Ratio, GGT, Fetuin-A) With Baseline-Adjusted Changes in Metabolic Factors From 1 to 3 Years Postpartum Baseline-Adjusted Changes Between 1 and 3 y Baseline-Adjusted Change in ALT Between 1 and 3 y Baseline-Adjusted Change in AST Between 1 and 3 y Baseline-Adjusted Change in ALT/AST Ratio Between 1 and 3 y Baseline-Adjusted Change in GGT Between 1 and 3 y Baseline-Adjusted Change in Fetuin-A Between 1 and 3 y r P r P r P r P r P BMI 0.10 0.10 0.02 0.78 0.11 0.06 0.22 0.0003 0.06 0.33 Waist 0.15 0.01 0.05 0.42 0.17 0.006 0.13 0.03 0.04 0.53 Matsuda index −0.06 0.31 0.16 0.007 −0.20 0.001 −0.29 <0.0001 −0.24 <0.0001 HOMA-IR 0.07 0.22 −0.12 0.04 0.21 0.0005 0.20 0.0009 0.20 0.0005 Insulinogenic index/HOMA-IR −0.03 0.69 0.04 0.55 −0.08 0.23 −0.06 0.31 −0.11 0.07 ISSI-2 −0.10 0.08 −0.03 0.58 −0.10 0.10 −0.07 0.24 0.01 0.85 Fasting glucose 0.08 0.19 −0.01 0.82 0.09 0.15 0.12 0.04 0.06 0.30 Baseline-Adjusted Changes Between 1 and 3 y Baseline-Adjusted Change in ALT Between 1 and 3 y Baseline-Adjusted Change in AST Between 1 and 3 y Baseline-Adjusted Change in ALT/AST Ratio Between 1 and 3 y Baseline-Adjusted Change in GGT Between 1 and 3 y Baseline-Adjusted Change in Fetuin-A Between 1 and 3 y r P r P r P r P r P BMI 0.10 0.10 0.02 0.78 0.11 0.06 0.22 0.0003 0.06 0.33 Waist 0.15 0.01 0.05 0.42 0.17 0.006 0.13 0.03 0.04 0.53 Matsuda index −0.06 0.31 0.16 0.007 −0.20 0.001 −0.29 <0.0001 −0.24 <0.0001 HOMA-IR 0.07 0.22 −0.12 0.04 0.21 0.0005 0.20 0.0009 0.20 0.0005 Insulinogenic index/HOMA-IR −0.03 0.69 0.04 0.55 −0.08 0.23 −0.06 0.31 −0.11 0.07 ISSI-2 −0.10 0.08 −0.03 0.58 −0.10 0.10 −0.07 0.24 0.01 0.85 Fasting glucose 0.08 0.19 −0.01 0.82 0.09 0.15 0.12 0.04 0.06 0.30 Bold indicates P < 0.05. View Large Table 2. Partial Spearman Correlations of Baseline-Adjusted Changes in Hepatic Markers (ALT, AST, ALT/AST Ratio, GGT, Fetuin-A) With Baseline-Adjusted Changes in Metabolic Factors From 1 to 3 Years Postpartum Baseline-Adjusted Changes Between 1 and 3 y Baseline-Adjusted Change in ALT Between 1 and 3 y Baseline-Adjusted Change in AST Between 1 and 3 y Baseline-Adjusted Change in ALT/AST Ratio Between 1 and 3 y Baseline-Adjusted Change in GGT Between 1 and 3 y Baseline-Adjusted Change in Fetuin-A Between 1 and 3 y r P r P r P r P r P BMI 0.10 0.10 0.02 0.78 0.11 0.06 0.22 0.0003 0.06 0.33 Waist 0.15 0.01 0.05 0.42 0.17 0.006 0.13 0.03 0.04 0.53 Matsuda index −0.06 0.31 0.16 0.007 −0.20 0.001 −0.29 <0.0001 −0.24 <0.0001 HOMA-IR 0.07 0.22 −0.12 0.04 0.21 0.0005 0.20 0.0009 0.20 0.0005 Insulinogenic index/HOMA-IR −0.03 0.69 0.04 0.55 −0.08 0.23 −0.06 0.31 −0.11 0.07 ISSI-2 −0.10 0.08 −0.03 0.58 −0.10 0.10 −0.07 0.24 0.01 0.85 Fasting glucose 0.08 0.19 −0.01 0.82 0.09 0.15 0.12 0.04 0.06 0.30 Baseline-Adjusted Changes Between 1 and 3 y Baseline-Adjusted Change in ALT Between 1 and 3 y Baseline-Adjusted Change in AST Between 1 and 3 y Baseline-Adjusted Change in ALT/AST Ratio Between 1 and 3 y Baseline-Adjusted Change in GGT Between 1 and 3 y Baseline-Adjusted Change in Fetuin-A Between 1 and 3 y r P r P r P r P r P BMI 0.10 0.10 0.02 0.78 0.11 0.06 0.22 0.0003 0.06 0.33 Waist 0.15 0.01 0.05 0.42 0.17 0.006 0.13 0.03 0.04 0.53 Matsuda index −0.06 0.31 0.16 0.007 −0.20 0.001 −0.29 <0.0001 −0.24 <0.0001 HOMA-IR 0.07 0.22 −0.12 0.04 0.21 0.0005 0.20 0.0009 0.20 0.0005 Insulinogenic index/HOMA-IR −0.03 0.69 0.04 0.55 −0.08 0.23 −0.06 0.31 −0.11 0.07 ISSI-2 −0.10 0.08 −0.03 0.58 −0.10 0.10 −0.07 0.24 0.01 0.85 Fasting glucose 0.08 0.19 −0.01 0.82 0.09 0.15 0.12 0.04 0.06 0.30 Bold indicates P < 0.05. View Large Table 3. Hepatic Markers at 1 Year and Their Changes From 1 to 3 Years as Predictors of the Matsuda Index at 3 Years Postpartum Model/Hepatic Markers β t P I  ALT at 1 y −0.003 −0.78 0.44  Change in ALT from 1 to 3 y −0.01 −2.61 0.01 II  AST at 1 y 0.002 0.28 0.78  Change in AST from 1 to 3 y 0.02 3.23 0.001 III  ALT/AST ratio at 1 y −0.24 −1.99 0.05  Change in ALT/AST ratio from 1 to 3 y −0.59 −5.47 <0.0001 IV  GGT at 1 y −0.004 −1.42 0.16  Change in GGT from 1 to 3 y −0.008 −3.20 0.002 V  Fetuin-A at 1 y −0.47 −3.17 0.002  Change in fetuin-A from 1 to 3 y −0.40 −3.56 0.0004 Model/Hepatic Markers β t P I  ALT at 1 y −0.003 −0.78 0.44  Change in ALT from 1 to 3 y −0.01 −2.61 0.01 II  AST at 1 y 0.002 0.28 0.78  Change in AST from 1 to 3 y 0.02 3.23 0.001 III  ALT/AST ratio at 1 y −0.24 −1.99 0.05  Change in ALT/AST ratio from 1 to 3 y −0.59 −5.47 <0.0001 IV  GGT at 1 y −0.004 −1.42 0.16  Change in GGT from 1 to 3 y −0.008 −3.20 0.002 V  Fetuin-A at 1 y −0.47 −3.17 0.002  Change in fetuin-A from 1 to 3 y −0.40 −3.56 0.0004 Five multiple linear regression models were constructed to evaluate the following hepatic markers at 1 y and their changes from 1 to 3 y: ALT (model I), AST (model II), ALT/AST ratio (model III), GGT (model IV), and fetuin-A (model V). Each model was adjusted for age, ethnicity, family history of T2DM, BMI at 1 y, change in BMI from 1 to 3 y, breastfeeding, and the Matsuda index at 1 y. View Large Table 3. Hepatic Markers at 1 Year and Their Changes From 1 to 3 Years as Predictors of the Matsuda Index at 3 Years Postpartum Model/Hepatic Markers β t P I  ALT at 1 y −0.003 −0.78 0.44  Change in ALT from 1 to 3 y −0.01 −2.61 0.01 II  AST at 1 y 0.002 0.28 0.78  Change in AST from 1 to 3 y 0.02 3.23 0.001 III  ALT/AST ratio at 1 y −0.24 −1.99 0.05  Change in ALT/AST ratio from 1 to 3 y −0.59 −5.47 <0.0001 IV  GGT at 1 y −0.004 −1.42 0.16  Change in GGT from 1 to 3 y −0.008 −3.20 0.002 V  Fetuin-A at 1 y −0.47 −3.17 0.002  Change in fetuin-A from 1 to 3 y −0.40 −3.56 0.0004 Model/Hepatic Markers β t P I  ALT at 1 y −0.003 −0.78 0.44  Change in ALT from 1 to 3 y −0.01 −2.61 0.01 II  AST at 1 y 0.002 0.28 0.78  Change in AST from 1 to 3 y 0.02 3.23 0.001 III  ALT/AST ratio at 1 y −0.24 −1.99 0.05  Change in ALT/AST ratio from 1 to 3 y −0.59 −5.47 <0.0001 IV  GGT at 1 y −0.004 −1.42 0.16  Change in GGT from 1 to 3 y −0.008 −3.20 0.002 V  Fetuin-A at 1 y −0.47 −3.17 0.002  Change in fetuin-A from 1 to 3 y −0.40 −3.56 0.0004 Five multiple linear regression models were constructed to evaluate the following hepatic markers at 1 y and their changes from 1 to 3 y: ALT (model I), AST (model II), ALT/AST ratio (model III), GGT (model IV), and fetuin-A (model V). Each model was adjusted for age, ethnicity, family history of T2DM, BMI at 1 y, change in BMI from 1 to 3 y, breastfeeding, and the Matsuda index at 1 y. View Large Table 4. Hepatic Markers at 1 Year and Their Changes From 1 to 3 Years as Predictors of IGI/HOMA-IR at 3 Years Postpartum Model/Hepatic Markers β t P I  ALT at 1 y −0.003 −0.31 0.75  Change in ALT from 1 to 3 y −0.01 −1.78 0.08 II  AST at 1 y 0.02 1.90 0.06  Change in AST from 1 to 3 y 0.01 1.52 0.13 III  ALT/AST ratio at 1 y −0.59 −2.45 0.01  Change in ALT/AST ratio from 1 to 3 y −0.73 −3.35 0.001 IV  GGT at 1 y 0.0006 0.12 0.90  Change in GGT from 1 to 3 y −0.002 −0.39 0.70 V  Fetuin-A at 1 y −0.26 −0.85 0.40  Change in fetuin-A from 1 to 3 y −0.25 −1.08 0.28 Model/Hepatic Markers β t P I  ALT at 1 y −0.003 −0.31 0.75  Change in ALT from 1 to 3 y −0.01 −1.78 0.08 II  AST at 1 y 0.02 1.90 0.06  Change in AST from 1 to 3 y 0.01 1.52 0.13 III  ALT/AST ratio at 1 y −0.59 −2.45 0.01  Change in ALT/AST ratio from 1 to 3 y −0.73 −3.35 0.001 IV  GGT at 1 y 0.0006 0.12 0.90  Change in GGT from 1 to 3 y −0.002 −0.39 0.70 V  Fetuin-A at 1 y −0.26 −0.85 0.40  Change in fetuin-A from 1 to 3 y −0.25 −1.08 0.28 Five multiple linear regression models were constructed to evaluate the following hepatic markers at 1 y and their changes from 1 to 3 y: ALT (model I), AST (model II), ALT/AST ratio (model III), GGT (model IV), and fetuin-A (model V). Each model was adjusted for age, ethnicity, family history of T2DM, BMI at 1 y, change in BMI from 1 to 3 y, breastfeeding, and IGI/HOMA-IR at 1 y. View Large Table 4. Hepatic Markers at 1 Year and Their Changes From 1 to 3 Years as Predictors of IGI/HOMA-IR at 3 Years Postpartum Model/Hepatic Markers β t P I  ALT at 1 y −0.003 −0.31 0.75  Change in ALT from 1 to 3 y −0.01 −1.78 0.08 II  AST at 1 y 0.02 1.90 0.06  Change in AST from 1 to 3 y 0.01 1.52 0.13 III  ALT/AST ratio at 1 y −0.59 −2.45 0.01  Change in ALT/AST ratio from 1 to 3 y −0.73 −3.35 0.001 IV  GGT at 1 y 0.0006 0.12 0.90  Change in GGT from 1 to 3 y −0.002 −0.39 0.70 V  Fetuin-A at 1 y −0.26 −0.85 0.40  Change in fetuin-A from 1 to 3 y −0.25 −1.08 0.28 Model/Hepatic Markers β t P I  ALT at 1 y −0.003 −0.31 0.75  Change in ALT from 1 to 3 y −0.01 −1.78 0.08 II  AST at 1 y 0.02 1.90 0.06  Change in AST from 1 to 3 y 0.01 1.52 0.13 III  ALT/AST ratio at 1 y −0.59 −2.45 0.01  Change in ALT/AST ratio from 1 to 3 y −0.73 −3.35 0.001 IV  GGT at 1 y 0.0006 0.12 0.90  Change in GGT from 1 to 3 y −0.002 −0.39 0.70 V  Fetuin-A at 1 y −0.26 −0.85 0.40  Change in fetuin-A from 1 to 3 y −0.25 −1.08 0.28 Five multiple linear regression models were constructed to evaluate the following hepatic markers at 1 y and their changes from 1 to 3 y: ALT (model I), AST (model II), ALT/AST ratio (model III), GGT (model IV), and fetuin-A (model V). Each model was adjusted for age, ethnicity, family history of T2DM, BMI at 1 y, change in BMI from 1 to 3 y, breastfeeding, and IGI/HOMA-IR at 1 y. View Large Table 5. Hepatic Markers at 1 Year and Their Changes From 1 to 3 Years as Predictors of Fasting Glucose at 3 Years Postpartum Model/Hepatic Markers β t P I  ALT at 1 y 0.004 1.01 0.31  Change in ALT from 1 to 3 y 0.007 1.98 0.05 II  AST at 1 y −0.003 −0.55 0.59  Change in AST from 1 to 3 y −0.002 −0.45 0.65 III  ALT/AST ratio at 1 y 0.20 1.62 0.11  Change in ALT/AST ratio from 1 to 3 y 0.29 2.55 0.01 IV  GGT at 1 y −0.003 −1.17 0.24  Change in GGT from 1 to 3 y 0.0008 0.32 0.75 V  Fetuin-A at 1 y −0.08 −0.53 0.59  Change in fetuin-A from 1 to 3 y 0.01 0.10 0.92 Model/Hepatic Markers β t P I  ALT at 1 y 0.004 1.01 0.31  Change in ALT from 1 to 3 y 0.007 1.98 0.05 II  AST at 1 y −0.003 −0.55 0.59  Change in AST from 1 to 3 y −0.002 −0.45 0.65 III  ALT/AST ratio at 1 y 0.20 1.62 0.11  Change in ALT/AST ratio from 1 to 3 y 0.29 2.55 0.01 IV  GGT at 1 y −0.003 −1.17 0.24  Change in GGT from 1 to 3 y 0.0008 0.32 0.75 V  Fetuin-A at 1 y −0.08 −0.53 0.59  Change in fetuin-A from 1 to 3 y 0.01 0.10 0.92 Five multiple linear regression models were constructed to evaluate the following hepatic markers at 1 y and their changes from 1 to 3 y: ALT (model I), AST (model II), ALT/AST ratio (model III), GGT (model IV), and fetuin-A (model V). Each model was adjusted for age, ethnicity, family history of T2DM, BMI at 1 y, change in BMI from 1 to 3 y, breastfeeding, and fasting glucose at 1 y. View Large Table 5. Hepatic Markers at 1 Year and Their Changes From 1 to 3 Years as Predictors of Fasting Glucose at 3 Years Postpartum Model/Hepatic Markers β t P I  ALT at 1 y 0.004 1.01 0.31  Change in ALT from 1 to 3 y 0.007 1.98 0.05 II  AST at 1 y −0.003 −0.55 0.59  Change in AST from 1 to 3 y −0.002 −0.45 0.65 III  ALT/AST ratio at 1 y 0.20 1.62 0.11  Change in ALT/AST ratio from 1 to 3 y 0.29 2.55 0.01 IV  GGT at 1 y −0.003 −1.17 0.24  Change in GGT from 1 to 3 y 0.0008 0.32 0.75 V  Fetuin-A at 1 y −0.08 −0.53 0.59  Change in fetuin-A from 1 to 3 y 0.01 0.10 0.92 Model/Hepatic Markers β t P I  ALT at 1 y 0.004 1.01 0.31  Change in ALT from 1 to 3 y 0.007 1.98 0.05 II  AST at 1 y −0.003 −0.55 0.59  Change in AST from 1 to 3 y −0.002 −0.45 0.65 III  ALT/AST ratio at 1 y 0.20 1.62 0.11  Change in ALT/AST ratio from 1 to 3 y 0.29 2.55 0.01 IV  GGT at 1 y −0.003 −1.17 0.24  Change in GGT from 1 to 3 y 0.0008 0.32 0.75 V  Fetuin-A at 1 y −0.08 −0.53 0.59  Change in fetuin-A from 1 to 3 y 0.01 0.10 0.92 Five multiple linear regression models were constructed to evaluate the following hepatic markers at 1 y and their changes from 1 to 3 y: ALT (model I), AST (model II), ALT/AST ratio (model III), GGT (model IV), and fetuin-A (model V). Each model was adjusted for age, ethnicity, family history of T2DM, BMI at 1 y, change in BMI from 1 to 3 y, breastfeeding, and fasting glucose at 1 y. View Large Finally, we conducted forward selection logistic regression analyses to determine independent predictors of prediabetes/diabetes at 3 years [defined as IFG, IGT, combined IFG/IGT, or diabetes, as per CDA guidelines (13)]. The covariates for selection were as follows: (1) diabetes risk factors (age, ethnicity, family history of diabetes, BMI at 1 year, change in BMI from 1 to 3 years, duration of breastfeeding), (2) glucose tolerance status at 1 year, and (3) all of the hepatic markers at 1 year (ALT, AST, GGT, fetuin-A) and the change in each hepatic marker from 1 to 3 years (Fig. 1). Age, ethnicity, family history of diabetes, BMI at 1 year, and duration of breastfeeding were forced into the model as major clinical risk factors for diabetes. The forward selection model was repeated with the inclusion of ALT/AST ratio and its change, in place of ALT and AST and their changes. Figure 1. View largeDownload slide Forward selection logistic regression model of (outcome) prediabetes/diabetes at 3 y postpartum, with the following covariates available for selection: age, ethnicity, family history of diabetes, BMI at 1 y, change in BMI from 1 to 3 y, duration of breastfeeding, glucose tolerance status at 1 y, all of the hepatic markers at 1 y (ALT, AST, GGT, and fetuin-A), and change in each hepatic marker from 1 to 3 y. Major clinical risk factors for diabetes (age, ethnicity, family history of diabetes, BMI at 1 y, and duration of breastfeeding) were forced into the model. Figure 1. View largeDownload slide Forward selection logistic regression model of (outcome) prediabetes/diabetes at 3 y postpartum, with the following covariates available for selection: age, ethnicity, family history of diabetes, BMI at 1 y, change in BMI from 1 to 3 y, duration of breastfeeding, glucose tolerance status at 1 y, all of the hepatic markers at 1 y (ALT, AST, GGT, and fetuin-A), and change in each hepatic marker from 1 to 3 y. Major clinical risk factors for diabetes (age, ethnicity, family history of diabetes, BMI at 1 y, and duration of breastfeeding) were forced into the model. Results Table 1 shows the characteristics of the study population at 1 year and 3 years postpartum, stratified into the following four groups based on their preceding gestational glucose tolerance status: (1) normal GCT NGT (n = 75), (2) abnormal GCT NGT (n = 98), (3) GIGT (n = 59), and (4) GDM (n = 104). At 1 year postpartum, the groups did not differ in age, ethnicity, family history of diabetes, duration of breastfeeding, smoking status, or BMI. As expected, at 1 year postpartum, there was a progressive decrease in insulin sensitivity (Matsuda index: P = 0.003) and β-cell function (IGI/HOMA-IR: P < 0.0001) from normal GCT NGT to abnormal GCT NGT to GIGT to GDM. Accordingly, there was a concomitant progressive rise in fasting glucose and 2-hour glucose across the four groups (both P < 0.0001), coupled with a stepwise increase in the prevalence of dysglycemia at 1 year from 2.8% to 12.8% to 22.2% to 29.6% (P < 0.0001), most of which (95%) was prediabetes. Of note, the hepatic markers ALT, AST, ALT/AST ratio, GGT, and fetuin-A did not differ across groups. At 3 years postpartum, the same patterns were apparent across the four gestational glucose tolerance groups for insulin sensitivity, β-cell function, fasting glucose, 2-hour glucose, and current glucose tolerance status. As before, the hepatic markers did not differ among the groups at 3 years. However, the change in ALT/AST ratio from 1 to 3 years differed across the groups (P = 0.05), with normal GCT NGT showing a smaller change than the three gestational dysglycemia groups. Changes in the other hepatic markers did not differ across the groups. After adjustment for diabetes risk factors (age, ethnicity, family history of diabetes, BMI, duration of breastfeeding) and current glucose tolerance, there were no statistically significant differences between the four gestational glucose tolerance groups in mean adjusted levels of any of the hepatic markers at 3 years (data not shown). Thus, between 1 and 3 years postpartum, the interval change in ALT/AST ratio was the only apparent hepatic marker difference between these four groups that reflected different degrees of future diabetic risk. Changes in hepatic markers and metabolic outcomes over time We next performed Spearman partial correlation analyses to evaluate the relationships between baseline-adjusted changes in hepatic markers and baseline-adjusted changes in metabolic factors from 1 to 3 years postpartum (Table 2). These analyses revealed that baseline-adjusted changes in ALT/AST ratio, GGT, and fetuin-A were inversely associated with baseline-adjusted changes in the Matsuda index, suggesting that increasing concentrations of these hepatic markers over time were accompanied by worsening insulin sensitivity. We next performed multiple linear regression analyses to determine whether any of the hepatic markers or their changes over time were independent predictors of the Matsuda index at 3 years, after adjustment for diabetes risk factors (age, ethnicity, family history of T2DM, breastfeeding, BMI at 1 year, change in BMI from 1 to 3 years) and the Matsuda index at 1 year (Table 3). Importantly, for each hepatic marker, its respective change from 1 to 3 years was an independent predictor of insulin sensitivity (Matsuda) at 3 years (Table 3). These relationships were strongest for ALT/AST ratio (t = −5.47, P < 0.0001) and fetuin-A (t = −3.56, P < 0.0001), both of which also showed independent associations of their baseline measures at 1 year with lower insulin sensitivity at 3 years. Similarly, the baseline measures at 1 year and changes from 1 to 3 years in ALT/AST ratio and fetuin-A both emerged as independent predictors of HOMA-IR at 3 years (Supplemental Table 1). Using the same modeling approach, we performed similar multiple linear regression analyses of our primary and secondary measures of β-cell function in Table 4 and Supplemental Table 2, respectively. These analyses showed that an increase in ALT/AST ratio from 1 to 3 years predicted poorer β-cell function as measured by either IGI/HOMA-IR (t = −3.35, P = 0.001) (Table 4) or ISSI-2 (t = −2.33, P = 0.02) (Supplemental Table 2). Moreover, similar multiple linear regression analyses of fasting glucose revealed that the increase in ALT/AST ratio from 1 to 3 years predicted higher fasting glycemia at 3 years (t = 2.55, P = 0.01) (Table 5). To evaluate the robustness of the findings from these multiple linear regression models in Tables 3 to 5 and Supplemental Tables 1 to 2, we performed a series of sensitivity analyses. The findings were largely unchanged in sensitivity analyses in which waist circumference at 1 year and the change in waist circumference from 1 to 3 years were included in the models, in place of baseline BMI and its change, respectively (data not shown). To address the possibility of false discovery, we also applied the correction method of Storey and confirmed that the findings remained unchanged (data not shown). Thus, from all of these analyses, ALT/AST ratio and fetuin-A emerged as hepatic markers that predicted pathophysiologic determinants of diabetes. Hepatic markers and prediabetes/diabetes Finally, we performed logistic regression analyses to determine whether any of the hepatic markers or their changes over time were independent predictors of prediabetes/diabetes at 3 years (Fig. 1). In a forward selection analysis that included baseline ALT, AST, GGT, and fetuin-A and their respective changes from 1 to 3 years as potential covariates, fetuin-A at 1 year emerged as a significant predictor of prediabetes/diabetes at 3 years (OR, 1.38; 95% CI, 1.01 to 1.88), accompanied by glucose intolerance at 1 year (OR, 11.0; 95% CI, 5.03 to 24.14), BMI at 1 year (OR, 1.44; 95% CI, 1.00 to 2.06), and the change in BMI from 1 to 3 years (OR, 1.55; 95% CI, 1.09 to 2.22). Furthermore, on sensitivity analyses with inclusion of ALT/AST ratio in place of ALT and AST, these findings were unchanged, with fetuin-A at 1 year again predicting prediabetes/diabetes (OR, 1.38; 95% CI, 1.01 to 1.88) (data not shown). Discussion In this study, we demonstrate three main findings. First, at both 1 and 3 years postpartum, the hepatic markers ALT, AST, GGT, and fetuin-A did not differ across four recent gestational tolerance groups reflecting different degrees of future diabetic risk. However, the intervening change in the ALT/AST ratio did differ between the groups. Second, changes over time in hepatic markers, particularly the ALT/AST ratio and fetuin-A, tracked with changes in insulin sensitivity and β-cell function. Third, and most important, at this early point in the natural history of T2DM, the ALT/AST ratio and fetuin-A emerged as independent predictors of fasting glycemia and prediabetes/diabetes, respectively. Taken together, these findings support a pathophysiologic basis in the relationship between hepatic markers and subsequent risk of T2DM. Although previous reports have linked fatty liver with insulin resistance in women with a history of GDM (19–21), few studies have evaluated circulating hepatic markers and their implications for diabetic risk in this population. In a cross-sectional study at mean 8 to 9 months postpartum, Rottenkolber et al. (22) reported that circulating fetuin-A was higher in 96 women with recent GDM compared with 51 controls. Forbes et al. (20) found that ALT and GGT did not differ between 110 women with previous GDM and 113 without such a history, at a mean 6 to 7 years postpartum. However, limitations of the studies to date have included modest samples sizes, cross-sectional evaluation at a single point in time, and potential heterogeneity in comparators (i.e., categorized as non-GDM, with identification after delivery). In this context, strengths of the current study are the prospective ascertainment of gestational glucose tolerance, yielding a well-characterized cohort of 336 women across the full spectrum of gestational glucose tolerance (from normal to GDM), coupled with assessment of both hepatic markers and metabolic outcomes on two occasions 2 years apart. With this approach, we demonstrate that, at both 1 and 3 years postpartum, ALT, AST, GGT, and fetuin-A did not differ across four recent gestational glucose tolerance groups that reflect distinct degrees of future diabetic risk. This study design also made it possible to evaluate the changes in hepatic markers over 2 years in these risk groups. In this regard, we observed that the three groups with higher future diabetic risk (GDM, GIGT, abnormal GCT NGT) experienced a greater increase in the ALT/AST ratio between 1 and 3 years than did the group with the lowest such risk (normal GCT NGT). Although an increased AST/ALT ratio (De Ritis ratio) has long been discussed clinically as a marker of liver disease (23), there has been limited previous evaluation in relation to the risk of T2DM. In a recent cross-sectional analysis from the Korean National Health and Nutrition Examination Survey, Ko et al. (24) reported that a lower AST/ALT ratio within the physiological range (i.e., hence higher ALT/AST ratio) was associated with a greater likelihood of IFG and T2DM. Similarly, in a cross-sectional study of nonobese Japanese adults, the ALT/AST ratio was associated with insulin resistance, as measured by HOMA-IR (25). Interestingly, when the Insulin Resistance and Atherosclerosis Study reported that this ratio predicted metabolic syndrome, the only component disorder thereof with which it was significantly associated in a series of separate models was IFG (26). Against this background, our findings suggest that, in this study population that is very early in the natural history of T2DM, the ALT/AST ratio may amplify a signal of future diabetic risk that is not yet detectable with individual liver enzymes alone. In this context, our evaluation of hepatic makers and metabolic function at two points in time is particularly informative by enabling not only the assessment of cross-sectional and longitudinal associations between these exposures and outcomes but also the relationship between their respective changes over time. We show that, although changes in all of the liver markers related to changes in insulin sensitivity (Table 3), the strongest predictors thereof were ALT/AST ratio and fetuin-A. Moreover, an increase in the ALT/AST ratio was the only hepatic marker that independently predicted lower β-cell function at 3 years, and it did so for both β-cell measures (Table 4, Supplemental Table 2). Finally, consistent with its associations with insulin resistance and β-cell dysfunction, rising ALT/AST ratio emerged as an independent predictor of higher fasting glucose at this early point in the natural history of diabetes (Table 5). Few previous studies have evaluated serial measurements of liver enzymes in relation to diabetic risk, although a secondary case-control analysis of the West of Scotland Coronary Prevention Study found elevations in ALT at 18 months, 12 months, and 6 months prior to the diagnosis of diabetes in 86 middle-aged male participants (27). The current study extends this concept by showing that a rising ALT/AST ratio tracks with insulin resistance, β-cell dysfunction, and fasting glucose early in the natural history of diabetes in young women. Although it has generally been believed that hepatic fat underlies the association between liver enzymes and diabetic risk (8, 9), a growing body of evidence suggests that this mechanistic basis may not fully apply to fetuin-A. First, in vitro studies have shown that fetuin-A reversibly binds the insulin receptor tyrosine kinase in peripheral tissues and can thereby directly induce insulin resistance (28, 29). Second, the Multiethnic Study of Atherosclerosis recently reported that baseline fetuin-A independently predicted incident diabetes in women, after adjustment for liver fat content (measured by computed tomography) (7). Third, in the Nurses’ Health Study, the circulating fetuin-A concentration predicted the subsequent risk of T2DM even after adjustment for liver enzymes (ALT and GGT) (6). Our findings are consistent with these observations and extend this literature in two ways. First, we demonstrate that both baseline fetuin-A and its change from 1 to 3 years postpartum were independently associated with lower whole-body insulin sensitivity (Table 3). Second, fetuin-A at 1 year emerged as an independent predictor of prediabetes/diabetes at 3 years in a forward selection analysis that included all of the liver enzymes, both at baseline and their change over time. Of note, it did so in the absence of an independent association with fasting glycemia, consistent with previous observations suggesting that elevated fetuin-A is a feature of IGT but not IFG (30, 31). Overall, our findings link both ALT/AST ratio and fetuin-A to early dysglycemia in young women but support the concept that they have distinct mechanistic bases in this regard. A limitation of this study is that some factors that could affect liver enzymes have not been evaluated. These include maternal infections, diet, and lifestyle practices. However, the current analyses were adjusted for major diabetes risk factors (age, ethnicity, family history, BMI) and breastfeeding history [which has been shown to have sustained effects on insulin sensitivity and long-term diabetic risk (32)]. Another limitation is that insulin sensitivity and β-cell function were assessed with OGTT-based surrogate indices rather than clamp studies. However, their time-consuming and invasive nature would have made clamp studies difficult to complete on two occasions over 2 years in 336 new mothers. Moreover, the Matsuda index, HOMA-IR, ISSI-2, and IGI/HOMA-IR are validated measures that have been widely used in previous studies (10–12, 14–18), and the serial OGTTs on which they were determined made it possible to also assess glucose tolerance status. In conclusion, at both 1 and 3 years postpartum, ALT, AST, GGT, and fetuin-A did not differ across four recent gestational tolerance groups reflecting different degrees of future diabetic risk. However, the interval change in the ALT/AST ratio differed between these groups. Moreover, change in the ALT/AST ratio and fetuin-A tracked with changes in insulin sensitivity and β-cell function, thereby linking these hepatic markers to the pathologic determinants of T2DM. Most important, the ALT/AST ratio and fetuin-A emerged as independent predictors of fasting glycemia and prediabetes/diabetes, respectively. Taken together, these findings thus support a pathophysiologic basis in the relationship between changes in circulating hepatic markers and diabetic risk very early in the natural history of T2DM in women. Abbreviations: Abbreviations: ALT alanine aminotransferase AST aspartate aminotransferase BMI body mass index CDA Canadian Diabetes Association GCT glucose challenge test GDM gestational diabetes mellitus GGT γ-glutamyltransferase GIGT gestational impaired glucose tolerance HOMA-IR homeostasis model assessment of insulin resistance IFG impaired fasting glucose IGI insulinogenic index IGT impaired glucose tolerance ISSI-2 Insulin Secretion-Sensitivity Index–2 NDDG National Diabetes Data Group NGT normal glucose tolerance OGTT oral glucose tolerance test T2DM type 2 diabetes mellitus Acknowledgments Financial Support: This study was supported by operating grants from the Canadian Institutes of Health Research (CIHR)(MOP-84206) and Canadian Diabetes Association (CDA)(CDA-OG-3-15-4924-RR) to R.R. A.J.H. holds a Tier-II Canada Research Chair in Diabetes Epidemiology. B.Z. holds the Sam and Judy Pencer Family Chair in Diabetes Research at Mount Sinai Hospital and University of Toronto. R.R. is supported by a Heart and Stroke Foundation of Ontario Mid-Career Investigator Award and holds the Boehringer Ingelheim Chair in Beta-cell Preservation, Function and Regeneration at Mount Sinai Hospital. Author Contributions: R.R., A.J.H., P.W.C., M.S., and B.Z. designed and implemented the study. R.R. and C.Y. contributed to the analysis plan and interpretation of the data. C.Y. performed the statistical analyses. L.P. wrote the first draft. All authors critically revised the manuscript for important intellectual content. All authors approved the final manuscript. R.R. is guarantor, had full access to all of the data in the study, and takes responsibility for the integrity of the data and the accuracy of the data analysis. Disclosure Summary: The authors have nothing to disclose. References 1. Hanley AJ , Williams K , Festa A , Wagenknecht LE , D’Agostino RB Jr , Kempf J , Zinman B , Haffner SM ; Insulin Resistance Atherosclerosis Study . 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Nonalcoholic fatty liver disease is associated with an almost twofold increased risk of incident type 2 diabetes and metabolic syndrome: evidence from a systematic review and meta-analysis . J Gastroenterol Hepatol . 2016 ; 31 ( 5 ): 936 – 944 . 10. Retnakaran R . Glucose tolerance status in pregnancy: a window to the future risk of diabetes and cardiovascular disease in young women . Curr Diabetes Rev . 2009 ; 5 ( 4 ): 239 – 244 . 11. Retnakaran R , Qi Y , Sermer M , Connelly PW , Hanley AJ , Zinman B . Glucose intolerance in pregnancy and future risk of pre-diabetes or diabetes . Diabetes Care . 2008 ; 31 ( 10 ): 2026 – 2031 . 12. Kramer CK , Swaminathan B , Hanley AJ , Connelly PW , Sermer M , Zinman B , Retnakaran R . Each degree of glucose intolerance in pregnancy predicts distinct trajectories of β-cell function, insulin sensitivity, and glycemia in the first 3 years postpartum . Diabetes Care . 2014 ; 37 ( 12 ): 3262 – 3269 . 13. Canadian Diabetes Association Clinical Practice Guidelines Expert Committee . Definition, classification and diagnosis of diabetes, prediabetes and metabolic syndrome . Can J Diabetes . 2013 ; 37 ( Suppl 1 ): S8 – S11 . 14. Matsuda M , DeFronzo RA . Insulin sensitivity indices obtained from oral glucose tolerance testing: comparison with the euglycemic insulin clamp . Diabetes Care . 1999 ; 22 ( 9 ): 1462 – 1470 . 15. Matthews DR , Hosker JP , Rudenski AS , Naylor BA , Treacher DF , Turner RC . Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man . Diabetologia . 1985 ; 28 ( 7 ): 412 – 419 . 16. Kahn SE . The relative contributions of insulin resistance and beta-cell dysfunction to the pathophysiology of type 2 diabetes . Diabetologia . 2003 ; 46 ( 1 ): 3 – 19 . 17. Retnakaran R , Shen S , Hanley AJ , Vuksan V , Hamilton JK , Zinman B . Hyperbolic relationship between insulin secretion and sensitivity on oral glucose tolerance test . Obesity (Silver Spring) . 2008 ; 16 ( 8 ): 1901 – 1907 . 18. Retnakaran R , Qi Y , Goran MI , Hamilton JK . Evaluation of proposed oral disposition index measures in relation to the actual disposition index . Diabet Med . 2009 ; 26 ( 12 ): 1198 – 1203 . 19. Tiikkainen M , Tamminen M , Häkkinen AM , Bergholm R , Vehkavaara S , Halavaara J , Teramo K , Rissanen A , Yki-Järvinen H . Liver-fat accumulation and insulin resistance in obese women with previous gestational diabetes . Obes Res . 2002 ; 10 ( 9 ): 859 – 867 . 20. Forbes S , Taylor-Robinson SD , Patel N , Allan P , Walker BR , Johnston DG . Increased prevalence of non-alcoholic fatty liver disease in European women with a history of gestational diabetes . Diabetologia . 2011 ; 54 : 641 – 647 . 21. Foghsgaard S , Andreasen C , Vedtofte L , Andersen ES , Bahne E , Strandberg C , Buhl T , Holst JJ , Svare JA , Clausen TD , Mathiesen ER , Damm P , Gluud LL , Knop FK , Vilsbøll T . Nonalcoholic fatty liver disease is prevalent in women with prior gestational diabetes mellitus and independently associated with insulin resistance and waist circumference . Diabetes Care . 2016 ; 40 ( 1 ): 109 – 116 . 22. Rottenkolber M , Ferrari U , Holland L , Aertsen S , Kammer NN , Hetterich H , Fugmann M , Banning F , Weise M , Sacco V , Kohn D , Freibothe I , Hutter S , Hasbargen U , Lehmann R , Grallert H , Parhofer KG , Seissler J , Lechner A . The diabetes risk phenotype of young women with recent gestational diabetes . J Clin Endocrinol Metab . 2015 ; 100 ( 6 ): E910 – E918 . 23. Botros M , Sikaris KA . The de Ritis ratio: the test of time . Clin Biochem Rev . 2013 ; 34 ( 3 ): 117 – 130 . 24. Ko SH , Baeg MK , Han KD , Ko SH , Ahn YB . Increased liver markers are associated with higher risk of type 2 diabetes . World J Gastroenterol . 2015 ; 21 ( 24 ): 7478 – 7487 . 25. Kawamoto R , Kohara K , Kusunoki T , Tabara Y , Abe M , Miki T . Alanine aminotransferase/aspartate aminotransferase ratio is the best surrogate marker for insulin resistance in non-obese Japanese adults . Cardiovasc Diabetol . 2012 ; 11 ( 1 ): 117 . 26. Hanley AJ , Williams K , Festa A , Wagenknecht LE , D’Agostino RB Jr , Haffner SM . Liver markers and development of the metabolic syndrome: the insulin resistance atherosclerosis study . Diabetes . 2005 ; 54 ( 11 ): 3140 – 3147 . 27. Sattar N , McConnachie A , Ford I , Gaw A , Cleland SJ , Forouhi NG , McFarlane P , Shepherd J , Cobbe S , Packard C . Serial metabolic measurements and conversion to type 2 diabetes in the west of Scotland coronary prevention study: specific elevations in alanine aminotransferase and triglycerides suggest hepatic fat accumulation as a potential contributing factor . Diabetes . 2007 ; 56 ( 4 ): 984 – 991 . 28. Rauth G , Pöschke O , Fink E , Eulitz M , Tippmer S , Kellerer M , Häring HU , Nawratil P , Haasemann M , Jahnen-Dechent W , Muller-Esterl W . The nucleotide and partial amino acid sequences of rat fetuin: identity with the natural tyrosine kinase inhibitor of the rat insulin receptor . Eur J Biochem . 1992 ; 204 ( 2 ): 523 – 529 . 29. Mathews ST , Srinivas PR , Leon MA , Grunberger G . Bovine fetuin is an inhibitor of insulin receptor tyrosine kinase . Life Sci . 1997 ; 61 ( 16 ): 1583 – 1592 . 30. Ou HY , Yang YC , Wu HT , Wu JS , Lu FH , Chang CJ . Increased fetuin-A concentrations in impaired glucose tolerance with or without nonalcoholic fatty liver disease, but not impaired fasting glucose . J Clin Endocrinol Metab . 2012 ; 97 ( 12 ): 4717 – 4723 . 31. Laughlin GA , Barrett-Connor E , Cummins KM , Daniels LB , Wassel CL , Ix JH . Sex-specific association of fetuin-A with type 2 diabetes in older community-dwelling adults: the Rancho Bernardo study . Diabetes Care . 2013 ; 36 ( 7 ): 1994 – 2000 . 32. Bajaj H , Ye C , Hanley AJ , Connelly PW , Sermer M , Zinman B , Retnakaran R . Prior lactation reduces future diabetic risk through sustained postweaning effects on insulin sensitivity . Am J Physiol Endocrinol Metab . 2017 ; 312 ( 3 ): E215 – E223 . Copyright © 2018 Endocrine Society http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Clinical Endocrinology and Metabolism Oxford University Press

Changes Over Time in Hepatic Markers Predict Changes in Insulin Sensitivity, β-Cell Function, and Glycemia

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Endocrine Society
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Copyright © 2018 Endocrine Society
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0021-972X
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1945-7197
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10.1210/jc.2018-00306
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Abstract

Abstract Context Serum concentrations of liver enzymes and the hepatokine fetuin-A have been linked to the risk of type 2 diabetes, but their longitudinal impact on insulin resistance and β-cell dysfunction is unclear. Objective To evaluate the impact of changes over 2 years in fetuin-A and the liver enzymes alanine aminotransferase (ALT), aspartate aminotransferase (AST), and γ-glutamyltransferase (GGT) on changes in insulin sensitivity, β-cell function, and glycemia in women with varying degrees of previous gestational dysglycemia, reflecting a range of future diabetic risk. Design/Setting/Participants In total, 336 women underwent glucose challenge test (GCT) and oral glucose tolerance test (OGTT) in pregnancy, followed by repeat OGTT and measurement of ALT/AST/GGT/fetuin-A at both 1 year and 3 years postpartum. The antepartum GCT/OGTT identified four gestational glucose tolerance groups: gestational diabetes (n = 104), gestational impaired glucose tolerance (n = 59), abnormal GCT with normal OGTT (n = 98), and normal GCT/OGTT (n = 75). Results At 1 and 3 years postpartum, ALT, AST, GGT, and fetuin-A did not differ across the four groups, but the intervening change in ALT/AST ratio was greater in the gestational dysglycemia groups (P = 0.05). Higher baseline ALT/AST (t = −1.99, P = 0.05) and fetuin-A (t = −3.17, P = 0.002) predicted lower insulin sensitivity (Matsuda) at 3 years, as did their respective changes from 1 to 3 years (ALT/AST: t = −5.47, P < 0.0001; fetuin-A: t = −3.56, P = 0.0004). Change in ALT/AST predicted lower β-cell function (t = −2.33, P = 0.02) and higher fasting glucose at 3 years (t = 2.55, P = 0.01). Moreover, baseline fetuin-A predicted prediabetes/diabetes at 3 years (OR, 1.38; 95% CI, 1.01 to 1.88). Conclusion Circulating hepatic markers, particularly ALT/AST ratio and fetuin-A, track with changes in insulin sensitivity and β-cell function, supporting a pathophysiologic basis in their prediction of diabetic risk. Over the past 15 years, several large prospective epidemiologic studies have shown that modest elevations of serum liver enzymes, such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), and γ-glutamyl transferase (GGT), are associated with an increased risk of subsequently developing type 2 diabetes (T2DM) (1–3). More recently, similar associations with incident T2DM have been reported with fetuin-A, a novel serum glycoprotein that is secreted by the liver into the circulation at high concentration (a “hepatokine”) (4–7). Collectively, it is believed that perturbations of these markers may be reflecting hepatic fat, the deposition of which is often enhanced in those who develop T2DM (8, 9). However, despite an abundance of epidemiologic studies in which baseline measurement of these hepatic markers has been associated with the subsequent development of T2DM, evidence supporting causality in this regard is limited. Notably, there is a relative paucity of human data linking changes in hepatic markers with changes in insulin sensitivity and β-cell function over time, as might be expected for causal mediators. Reasons for this evidence gap include (1) the fact that most studies have measured these markers at baseline only, (2) few have assessed insulin sensitivity and β-cell function, and (3) even fewer have evaluated changes over time in these factors. Thus, recognizing these limitations, we sought to evaluate the longitudinal relationship over time between hepatic markers and concurrent changes in insulin sensitivity and β-cell function in a population at risk for T2DM. Glucose intolerance in pregnancy provides the unique opportunity to identify just such a patient population. Indeed, each degree of antepartum glucose intolerance [ranging from gestational diabetes mellitus (GDM) to milder gestational impaired glucose tolerance (GIGT) to lesser dysglycemia to normoglycemia] identifies a proportionate level of future risk of T2DM (one that is highest in GDM, followed by GIGT, etc) (10–12). Moreover, each degree of gestational glucose intolerance predicts distinct trajectories of insulin sensitivity, β-cell function, and glycemia in the first 3 years postpartum (12). Accordingly, the early postpartum years following gestational dysglycemia can provide a model of the early natural history of T2DM with which to evaluate the impact of hepatic markers on glucose homeostasis. In this setting, we hypothesized that changes in hepatic makers would be associated with changes in insulin sensitivity and/or β-cell function. Thus, our objective in this study was to characterize the impact of changes between 1 and 3 years postpartum in liver enzymes (ALT, AST, GGT) and fetuin-A on changes in insulin sensitivity, β-cell function, and glycemia in women with varying degrees of previous gestational dysglycemia and hence a range of future diabetic risk. Methods The study population consisted of women participating in a prospective observational cohort study in which we are evaluating the relationship between glucose tolerance in pregnancy and metabolic function in the years after delivery. The study protocol has been described in detail (12). In brief, women are first recruited at the time of antepartum screening for GDM in the late second/early third trimester and undergo metabolic characterization at recruitment and again at 3 months and 1 year postpartum. At the latter visit, they are recruited into an ongoing long-term observational cohort study in which participating women undergo serial metabolic characterization biannually thereafter. This analysis reports on the associations of hepatic markers with metabolic function in 336 women who have completed their 3-year postpartum visit. The study protocol has been approved by the Mount Sinai Hospital Research Ethics Board, and all women have provided written informed consent for their participation. Recruitment and determination of glucose tolerance status in pregnancy At our institution, all pregnant women are screened for GDM by a 50-g glucose challenge test (GCT), followed by referral for a diagnostic oral glucose tolerance test (OGTT) if the GCT is abnormal (plasma glucose ≥7.8 mmol/L at 1 hour after ingestion of a 50-g glucose load). For this study, women are recruited either before or after the GCT, and all participants undergo a 3-hour 100-g OGTT for ascertainment of gestational glucose tolerance status, regardless of the GCT result (i.e., even if normal). As previously described (12), the recruitment of women after an abnormal GCT serves to enrich the study population for those with varying degrees of glucose intolerance. The GCT and OGTT enable stratification of participants into the following gestational glucose tolerance groups: (1) GDM, defined by National Diabetes Data Group (NDDG) criteria, which require at least two of the following on the OGTT: fasting blood glucose ≥5.8 mmol/L, 1-hour glucose ≥10.6 mmol/L, 2-hour glucose ≥9.2 mmol/L, or 3-hour glucose ≥8.1 mmol/L (2) GIGT, defined by meeting only one of the above NDDG criteria (3) Abnormal GCT with normal glucose tolerance (NGT), defined by an abnormal GCT followed by NGT on the OGTT (i.e., meeting none of the NDDG criteria) (4) Normal GCT NGT, defined by a normal GCT followed by NGT on the OGTT These four groups reflect the full spectrum of future diabetic risk and have been previously shown to predict distinct trajectories of insulin sensitivity, β-cell function, and glycemia in the first 3 years postpartum (12). Assessments at 1 and 3 years postpartum Participants return to the clinical investigation unit at 1 and 3 years for metabolic characterization, including 2-hour 75-g OGTT on both occasions (12). On each OGTT, current glucose tolerance status was defined according to Canadian Diabetes Association (CDA) guidelines (13). CDA guidelines define prediabetes as impaired glucose tolerance (IGT), impaired fasting glucose (IFG), or combined IFG/IGT (13). All OGTTs were performed in the morning after an overnight fast, with venous blood samples drawn for measurement of glucose and specific insulin at fasting and at 30, 60, and 120 minutes following ingestion of the glucose load. Specific insulin was measured with the Roche-Elecsys-1010 immunoassay analyzer and electrochemiluminescence immunoassay kit (Roche Diagnostics, Laval, QB, Canada). On each OGTT, the primary measure of insulin sensitivity was the Matsuda index (14), with the homeostasis model assessment of insulin resistance (HOMA-IR) providing a secondary measure (15). The primary measure of β-cell function was the insulinogenic index (IGI)/HOMA-IR (16), with the Insulin Secretion-Sensitivity Index–2 (ISSI-2) providing a secondary measure (17, 18). Fetuin-A was measured by an enzyme-linked immunosorbent assay (ALPCO, Salem, NH), with a lower limit of detection of 5.0 μg/L and an upper limit of detection of 216 μg/L. ALT, AST, and GGT were measured by the routine clinical biochemistry laboratory at Mount Sinai Hospital. Statistical analyses All analyses were conducted using SAS 9.2 (SAS Institute, Cary, NC). All tests were two-sided and performed at a significance level of P < 0.05. Characteristics of the gestational glucose tolerance groups were compared at 1 year and 3 years postpartum by either one-way analysis of variance or Kruskal-Wallis test for continuous variables, or either χ2 or Fisher exact test for categorical variables (Table 1). In particular, changes in hepatic markers from 1 to 3 years were compared between these groups (i.e., groups that reflect different degrees of diabetic risk). Table 1. Demographic, Clinical, and Metabolic Characteristics of Study Population at 1 Year and 3 Years Postpartum, Stratified Into the Following Four Groups Based on Gestational Glucose Tolerance Status: Normal GCT NGT, Abnormal GCT NGT, GIGT, and GDM Characteristic Normal GCT NGT (n = 75) Abnormal GCT NGT (n = 98) GIGT (n = 59) GDM (n = 104) P Value At 1 y postpartum  Age, y 36 ± 4 36 ± 4 36 ± 4 36 ± 4 0.82  Ethnicity, n (%) 0.50   White 57 (76.0) 73 (74.5) 40 (67.8) 69 (66.4)   Asian 5 (6.7) 9 (9.2) 7 (11.9) 17 (16.4)   Other 13 (17.3) 16 (16.3) 12 (20.3) 18 (17.3)  Family history of T2DM, n (%) 40 (53.3) 58 (59.2) 38 (64.4) 68 (65.4) 0.38  Months breastfeeding, mo 11 (6–12) 10 (6–12) 9 (4–12) 10 (3–12) 0.43  Current smoking, n (%) 2 (2.7) 3 (3.1) 5 (8.8) 2 (2.0) 0.21  BMI, kg/m2 24.4 (21.5–28.4) 23.7 (21.8–27.8) 25.4 (23.1–30.1) 25.5 (22.5–29.5) 0.12  Waist circumference, cm 87 ± 12 85 ± 12 89 ± 13 90 ± 14 0.04  Insulin sensitivity/resistance   Matsuda index 11.0 (6.4–15.5) 10.2 (6.1–13.8) 7.5 (4.9–12.9) 7.7 (4.4–11.0) 0.003   HOMA-IR 1.1 (0.7–1.7) 1.1 (0.6–1.7) 1.4 (0.8–2.0) 1.2 (0.7–2.0) 0.14  β-Cell function   ISSI-2 877 ± 292 841 ± 324 655 ± 227 673 ± 267 <0.0001   Insulinogenic index/HOMA-IR 13.7 (8.7–22.8) 10.6 (6.6–16.4) 7.0 (4.0–9.1) 7.3 (4.5–12.1) <0.0001  OGTT   Fasting glucose, mmol/L 4.6 ± 0.3 4.6 ± 0.4 4.9 ± 0.6 4.9 ± 0.5 <0.0001   2-h glucose, mmol/L 5.4 ± 1.2 6.1 ± 1.6 6.4 ± 1.8 6.9 ± 1.9 <0.0001  Current glucose tolerance, n (%) <0.0001   Normal 69 (97.2) 82 (87.2) 42 (77.8) 69 (70.4)   Prediabetes/diabetes 2 (2.8) 12 (12.8) 12 (22.2) 29 (29.6)  ALT, IU/L 11 (9–15) 11 (9–15) 12 (9–17) 12 (9–14) 0.81  AST, IU/L 18 (17–22) 19 (17–21) 19 (16–24) 19 (16–23) 0.97  ALT/AST ratio 0.71 ± 0.38 0.63 ± 0.21 0.65 ± 0.29 0.64 ± 0.28 0.41  GGT, IU/L 10 (8.0–15) 11 (8–14) 12 (8–17) 12 (8–17) 0.32  Fetuin-A, g/L 0.5 (0.4–0.5) 0.5 (0.4–0.6) 0.5 (0.4–0.6) 0.5 (0.4–0.6) 0.10 At 3 y postpartum  BMI, kg/m2 25.5 ± 4.5 25.4 ± 5.0 26.6 ± 4.8 26.9 ± 6.1 0.14  Waist circumference, cm 88 ± 12 86 ± 12 89 ± 12 90 ± 13 0.14  Insulin sensitivity/resistance   Matsuda index 10.0 (7.4–13.0) 8.2 (5.5–12.3) 7.3 (4.3–10.8) 6.2 (4.1–9.6) <0.0001   HOMA-IR 1.1 (0.7–1.8) 1.2 (0.7–1.9) 1.4 (1.0–2.1) 1.5 (1.0–2.4) 0.01  β-Cell function   ISSI-2 925 ± 331 898 ± 397 759 ± 333 668 ± 365 <0.0001   Insulinogenic index/HOMA-IR 13.1 (9.0–22.6) 10.7 (8.1–18.4) 8.7 (4.6–13.4) 6.8 (4.4–11.3) <0.0001  OGTT   Fasting glucose, mmol/L 4.6 ± 0.4 4.6 ± 0.5 4.8 ± 0.5 4.9 ± 0.6 <0.0001   2-h glucose, mmol/L 5.6 ± 1.2 6.1 ± 1.7 6.6 ± 2.1 7.4 ± 2.2 <0.0001  Current glucose tolerance, n (%) <0.0001   Normal 70 (93.3) 84 (85.7) 47 (80.0) 66 (63.5)   Prediabetes/diabetes 5 (6.7) 14 (14.3) 12 (20.0) 38 (36.5)  ALT, IU/L 15 (12–19) 15 (12–19) 15 (10–18) 16 (13–20) 0.69  AST, IU/L 18 (16–23) 18 (16–22) 17 (15–20) 18 (15–22) 0.34  ALT/AST ratio 0.86 ± 0.26 0.87 ± 0.28 0.90 ± 0.33 0.88 ± 0.22 0.80  GGT, IU/L 12 (9–17) 12 (10–17) 13 (10–18) 13 (11–22) 0.21  Fetuin-A, g/L 0.6 (0.5–0.7) 0.6 (0.5–0.8) 0.6 (0.5–0.8) 0.6 (0.5–0.7) 0.24 Changes from 1 to 3 y  Change in ALT 1.3 ± 11.7 4.4 ± 6.2 2.4 ± 10.7 3.8 ± 9.3 0.23  Change in AST −1.0 ± 8.6 −0.9 ± 5.5 −3.9 ± 8.1 −1.5 ± 7.5 0.11  Change in ALT/AST ratio 0.14 ± 0.36 0.26 ± 0.25 0.28 ± 0.32 0.25 ± 0.28 0.05  Change in GGT 0.8 ± 9.8 2.3 ± 7.1 4.6 ± 17.6 1.5 ± 12.6 0.38  Change in fetuin-A 0.1 ± 0.2 0.1 ± 0.3 0.1 ± 0.4 0.1 ± 0.3 0.98 Characteristic Normal GCT NGT (n = 75) Abnormal GCT NGT (n = 98) GIGT (n = 59) GDM (n = 104) P Value At 1 y postpartum  Age, y 36 ± 4 36 ± 4 36 ± 4 36 ± 4 0.82  Ethnicity, n (%) 0.50   White 57 (76.0) 73 (74.5) 40 (67.8) 69 (66.4)   Asian 5 (6.7) 9 (9.2) 7 (11.9) 17 (16.4)   Other 13 (17.3) 16 (16.3) 12 (20.3) 18 (17.3)  Family history of T2DM, n (%) 40 (53.3) 58 (59.2) 38 (64.4) 68 (65.4) 0.38  Months breastfeeding, mo 11 (6–12) 10 (6–12) 9 (4–12) 10 (3–12) 0.43  Current smoking, n (%) 2 (2.7) 3 (3.1) 5 (8.8) 2 (2.0) 0.21  BMI, kg/m2 24.4 (21.5–28.4) 23.7 (21.8–27.8) 25.4 (23.1–30.1) 25.5 (22.5–29.5) 0.12  Waist circumference, cm 87 ± 12 85 ± 12 89 ± 13 90 ± 14 0.04  Insulin sensitivity/resistance   Matsuda index 11.0 (6.4–15.5) 10.2 (6.1–13.8) 7.5 (4.9–12.9) 7.7 (4.4–11.0) 0.003   HOMA-IR 1.1 (0.7–1.7) 1.1 (0.6–1.7) 1.4 (0.8–2.0) 1.2 (0.7–2.0) 0.14  β-Cell function   ISSI-2 877 ± 292 841 ± 324 655 ± 227 673 ± 267 <0.0001   Insulinogenic index/HOMA-IR 13.7 (8.7–22.8) 10.6 (6.6–16.4) 7.0 (4.0–9.1) 7.3 (4.5–12.1) <0.0001  OGTT   Fasting glucose, mmol/L 4.6 ± 0.3 4.6 ± 0.4 4.9 ± 0.6 4.9 ± 0.5 <0.0001   2-h glucose, mmol/L 5.4 ± 1.2 6.1 ± 1.6 6.4 ± 1.8 6.9 ± 1.9 <0.0001  Current glucose tolerance, n (%) <0.0001   Normal 69 (97.2) 82 (87.2) 42 (77.8) 69 (70.4)   Prediabetes/diabetes 2 (2.8) 12 (12.8) 12 (22.2) 29 (29.6)  ALT, IU/L 11 (9–15) 11 (9–15) 12 (9–17) 12 (9–14) 0.81  AST, IU/L 18 (17–22) 19 (17–21) 19 (16–24) 19 (16–23) 0.97  ALT/AST ratio 0.71 ± 0.38 0.63 ± 0.21 0.65 ± 0.29 0.64 ± 0.28 0.41  GGT, IU/L 10 (8.0–15) 11 (8–14) 12 (8–17) 12 (8–17) 0.32  Fetuin-A, g/L 0.5 (0.4–0.5) 0.5 (0.4–0.6) 0.5 (0.4–0.6) 0.5 (0.4–0.6) 0.10 At 3 y postpartum  BMI, kg/m2 25.5 ± 4.5 25.4 ± 5.0 26.6 ± 4.8 26.9 ± 6.1 0.14  Waist circumference, cm 88 ± 12 86 ± 12 89 ± 12 90 ± 13 0.14  Insulin sensitivity/resistance   Matsuda index 10.0 (7.4–13.0) 8.2 (5.5–12.3) 7.3 (4.3–10.8) 6.2 (4.1–9.6) <0.0001   HOMA-IR 1.1 (0.7–1.8) 1.2 (0.7–1.9) 1.4 (1.0–2.1) 1.5 (1.0–2.4) 0.01  β-Cell function   ISSI-2 925 ± 331 898 ± 397 759 ± 333 668 ± 365 <0.0001   Insulinogenic index/HOMA-IR 13.1 (9.0–22.6) 10.7 (8.1–18.4) 8.7 (4.6–13.4) 6.8 (4.4–11.3) <0.0001  OGTT   Fasting glucose, mmol/L 4.6 ± 0.4 4.6 ± 0.5 4.8 ± 0.5 4.9 ± 0.6 <0.0001   2-h glucose, mmol/L 5.6 ± 1.2 6.1 ± 1.7 6.6 ± 2.1 7.4 ± 2.2 <0.0001  Current glucose tolerance, n (%) <0.0001   Normal 70 (93.3) 84 (85.7) 47 (80.0) 66 (63.5)   Prediabetes/diabetes 5 (6.7) 14 (14.3) 12 (20.0) 38 (36.5)  ALT, IU/L 15 (12–19) 15 (12–19) 15 (10–18) 16 (13–20) 0.69  AST, IU/L 18 (16–23) 18 (16–22) 17 (15–20) 18 (15–22) 0.34  ALT/AST ratio 0.86 ± 0.26 0.87 ± 0.28 0.90 ± 0.33 0.88 ± 0.22 0.80  GGT, IU/L 12 (9–17) 12 (10–17) 13 (10–18) 13 (11–22) 0.21  Fetuin-A, g/L 0.6 (0.5–0.7) 0.6 (0.5–0.8) 0.6 (0.5–0.8) 0.6 (0.5–0.7) 0.24 Changes from 1 to 3 y  Change in ALT 1.3 ± 11.7 4.4 ± 6.2 2.4 ± 10.7 3.8 ± 9.3 0.23  Change in AST −1.0 ± 8.6 −0.9 ± 5.5 −3.9 ± 8.1 −1.5 ± 7.5 0.11  Change in ALT/AST ratio 0.14 ± 0.36 0.26 ± 0.25 0.28 ± 0.32 0.25 ± 0.28 0.05  Change in GGT 0.8 ± 9.8 2.3 ± 7.1 4.6 ± 17.6 1.5 ± 12.6 0.38  Change in fetuin-A 0.1 ± 0.2 0.1 ± 0.3 0.1 ± 0.4 0.1 ± 0.3 0.98 P values are for overall comparison across groups by one-way analysis of variance or Kruskal-Wallis test for continuous variables, or either χ2 or Fisher exact test for categorical variables. Continuous variables are presented as mean ± SD (if normally distributed) or median with interquartile range (if skewed). View Large Table 1. Demographic, Clinical, and Metabolic Characteristics of Study Population at 1 Year and 3 Years Postpartum, Stratified Into the Following Four Groups Based on Gestational Glucose Tolerance Status: Normal GCT NGT, Abnormal GCT NGT, GIGT, and GDM Characteristic Normal GCT NGT (n = 75) Abnormal GCT NGT (n = 98) GIGT (n = 59) GDM (n = 104) P Value At 1 y postpartum  Age, y 36 ± 4 36 ± 4 36 ± 4 36 ± 4 0.82  Ethnicity, n (%) 0.50   White 57 (76.0) 73 (74.5) 40 (67.8) 69 (66.4)   Asian 5 (6.7) 9 (9.2) 7 (11.9) 17 (16.4)   Other 13 (17.3) 16 (16.3) 12 (20.3) 18 (17.3)  Family history of T2DM, n (%) 40 (53.3) 58 (59.2) 38 (64.4) 68 (65.4) 0.38  Months breastfeeding, mo 11 (6–12) 10 (6–12) 9 (4–12) 10 (3–12) 0.43  Current smoking, n (%) 2 (2.7) 3 (3.1) 5 (8.8) 2 (2.0) 0.21  BMI, kg/m2 24.4 (21.5–28.4) 23.7 (21.8–27.8) 25.4 (23.1–30.1) 25.5 (22.5–29.5) 0.12  Waist circumference, cm 87 ± 12 85 ± 12 89 ± 13 90 ± 14 0.04  Insulin sensitivity/resistance   Matsuda index 11.0 (6.4–15.5) 10.2 (6.1–13.8) 7.5 (4.9–12.9) 7.7 (4.4–11.0) 0.003   HOMA-IR 1.1 (0.7–1.7) 1.1 (0.6–1.7) 1.4 (0.8–2.0) 1.2 (0.7–2.0) 0.14  β-Cell function   ISSI-2 877 ± 292 841 ± 324 655 ± 227 673 ± 267 <0.0001   Insulinogenic index/HOMA-IR 13.7 (8.7–22.8) 10.6 (6.6–16.4) 7.0 (4.0–9.1) 7.3 (4.5–12.1) <0.0001  OGTT   Fasting glucose, mmol/L 4.6 ± 0.3 4.6 ± 0.4 4.9 ± 0.6 4.9 ± 0.5 <0.0001   2-h glucose, mmol/L 5.4 ± 1.2 6.1 ± 1.6 6.4 ± 1.8 6.9 ± 1.9 <0.0001  Current glucose tolerance, n (%) <0.0001   Normal 69 (97.2) 82 (87.2) 42 (77.8) 69 (70.4)   Prediabetes/diabetes 2 (2.8) 12 (12.8) 12 (22.2) 29 (29.6)  ALT, IU/L 11 (9–15) 11 (9–15) 12 (9–17) 12 (9–14) 0.81  AST, IU/L 18 (17–22) 19 (17–21) 19 (16–24) 19 (16–23) 0.97  ALT/AST ratio 0.71 ± 0.38 0.63 ± 0.21 0.65 ± 0.29 0.64 ± 0.28 0.41  GGT, IU/L 10 (8.0–15) 11 (8–14) 12 (8–17) 12 (8–17) 0.32  Fetuin-A, g/L 0.5 (0.4–0.5) 0.5 (0.4–0.6) 0.5 (0.4–0.6) 0.5 (0.4–0.6) 0.10 At 3 y postpartum  BMI, kg/m2 25.5 ± 4.5 25.4 ± 5.0 26.6 ± 4.8 26.9 ± 6.1 0.14  Waist circumference, cm 88 ± 12 86 ± 12 89 ± 12 90 ± 13 0.14  Insulin sensitivity/resistance   Matsuda index 10.0 (7.4–13.0) 8.2 (5.5–12.3) 7.3 (4.3–10.8) 6.2 (4.1–9.6) <0.0001   HOMA-IR 1.1 (0.7–1.8) 1.2 (0.7–1.9) 1.4 (1.0–2.1) 1.5 (1.0–2.4) 0.01  β-Cell function   ISSI-2 925 ± 331 898 ± 397 759 ± 333 668 ± 365 <0.0001   Insulinogenic index/HOMA-IR 13.1 (9.0–22.6) 10.7 (8.1–18.4) 8.7 (4.6–13.4) 6.8 (4.4–11.3) <0.0001  OGTT   Fasting glucose, mmol/L 4.6 ± 0.4 4.6 ± 0.5 4.8 ± 0.5 4.9 ± 0.6 <0.0001   2-h glucose, mmol/L 5.6 ± 1.2 6.1 ± 1.7 6.6 ± 2.1 7.4 ± 2.2 <0.0001  Current glucose tolerance, n (%) <0.0001   Normal 70 (93.3) 84 (85.7) 47 (80.0) 66 (63.5)   Prediabetes/diabetes 5 (6.7) 14 (14.3) 12 (20.0) 38 (36.5)  ALT, IU/L 15 (12–19) 15 (12–19) 15 (10–18) 16 (13–20) 0.69  AST, IU/L 18 (16–23) 18 (16–22) 17 (15–20) 18 (15–22) 0.34  ALT/AST ratio 0.86 ± 0.26 0.87 ± 0.28 0.90 ± 0.33 0.88 ± 0.22 0.80  GGT, IU/L 12 (9–17) 12 (10–17) 13 (10–18) 13 (11–22) 0.21  Fetuin-A, g/L 0.6 (0.5–0.7) 0.6 (0.5–0.8) 0.6 (0.5–0.8) 0.6 (0.5–0.7) 0.24 Changes from 1 to 3 y  Change in ALT 1.3 ± 11.7 4.4 ± 6.2 2.4 ± 10.7 3.8 ± 9.3 0.23  Change in AST −1.0 ± 8.6 −0.9 ± 5.5 −3.9 ± 8.1 −1.5 ± 7.5 0.11  Change in ALT/AST ratio 0.14 ± 0.36 0.26 ± 0.25 0.28 ± 0.32 0.25 ± 0.28 0.05  Change in GGT 0.8 ± 9.8 2.3 ± 7.1 4.6 ± 17.6 1.5 ± 12.6 0.38  Change in fetuin-A 0.1 ± 0.2 0.1 ± 0.3 0.1 ± 0.4 0.1 ± 0.3 0.98 Characteristic Normal GCT NGT (n = 75) Abnormal GCT NGT (n = 98) GIGT (n = 59) GDM (n = 104) P Value At 1 y postpartum  Age, y 36 ± 4 36 ± 4 36 ± 4 36 ± 4 0.82  Ethnicity, n (%) 0.50   White 57 (76.0) 73 (74.5) 40 (67.8) 69 (66.4)   Asian 5 (6.7) 9 (9.2) 7 (11.9) 17 (16.4)   Other 13 (17.3) 16 (16.3) 12 (20.3) 18 (17.3)  Family history of T2DM, n (%) 40 (53.3) 58 (59.2) 38 (64.4) 68 (65.4) 0.38  Months breastfeeding, mo 11 (6–12) 10 (6–12) 9 (4–12) 10 (3–12) 0.43  Current smoking, n (%) 2 (2.7) 3 (3.1) 5 (8.8) 2 (2.0) 0.21  BMI, kg/m2 24.4 (21.5–28.4) 23.7 (21.8–27.8) 25.4 (23.1–30.1) 25.5 (22.5–29.5) 0.12  Waist circumference, cm 87 ± 12 85 ± 12 89 ± 13 90 ± 14 0.04  Insulin sensitivity/resistance   Matsuda index 11.0 (6.4–15.5) 10.2 (6.1–13.8) 7.5 (4.9–12.9) 7.7 (4.4–11.0) 0.003   HOMA-IR 1.1 (0.7–1.7) 1.1 (0.6–1.7) 1.4 (0.8–2.0) 1.2 (0.7–2.0) 0.14  β-Cell function   ISSI-2 877 ± 292 841 ± 324 655 ± 227 673 ± 267 <0.0001   Insulinogenic index/HOMA-IR 13.7 (8.7–22.8) 10.6 (6.6–16.4) 7.0 (4.0–9.1) 7.3 (4.5–12.1) <0.0001  OGTT   Fasting glucose, mmol/L 4.6 ± 0.3 4.6 ± 0.4 4.9 ± 0.6 4.9 ± 0.5 <0.0001   2-h glucose, mmol/L 5.4 ± 1.2 6.1 ± 1.6 6.4 ± 1.8 6.9 ± 1.9 <0.0001  Current glucose tolerance, n (%) <0.0001   Normal 69 (97.2) 82 (87.2) 42 (77.8) 69 (70.4)   Prediabetes/diabetes 2 (2.8) 12 (12.8) 12 (22.2) 29 (29.6)  ALT, IU/L 11 (9–15) 11 (9–15) 12 (9–17) 12 (9–14) 0.81  AST, IU/L 18 (17–22) 19 (17–21) 19 (16–24) 19 (16–23) 0.97  ALT/AST ratio 0.71 ± 0.38 0.63 ± 0.21 0.65 ± 0.29 0.64 ± 0.28 0.41  GGT, IU/L 10 (8.0–15) 11 (8–14) 12 (8–17) 12 (8–17) 0.32  Fetuin-A, g/L 0.5 (0.4–0.5) 0.5 (0.4–0.6) 0.5 (0.4–0.6) 0.5 (0.4–0.6) 0.10 At 3 y postpartum  BMI, kg/m2 25.5 ± 4.5 25.4 ± 5.0 26.6 ± 4.8 26.9 ± 6.1 0.14  Waist circumference, cm 88 ± 12 86 ± 12 89 ± 12 90 ± 13 0.14  Insulin sensitivity/resistance   Matsuda index 10.0 (7.4–13.0) 8.2 (5.5–12.3) 7.3 (4.3–10.8) 6.2 (4.1–9.6) <0.0001   HOMA-IR 1.1 (0.7–1.8) 1.2 (0.7–1.9) 1.4 (1.0–2.1) 1.5 (1.0–2.4) 0.01  β-Cell function   ISSI-2 925 ± 331 898 ± 397 759 ± 333 668 ± 365 <0.0001   Insulinogenic index/HOMA-IR 13.1 (9.0–22.6) 10.7 (8.1–18.4) 8.7 (4.6–13.4) 6.8 (4.4–11.3) <0.0001  OGTT   Fasting glucose, mmol/L 4.6 ± 0.4 4.6 ± 0.5 4.8 ± 0.5 4.9 ± 0.6 <0.0001   2-h glucose, mmol/L 5.6 ± 1.2 6.1 ± 1.7 6.6 ± 2.1 7.4 ± 2.2 <0.0001  Current glucose tolerance, n (%) <0.0001   Normal 70 (93.3) 84 (85.7) 47 (80.0) 66 (63.5)   Prediabetes/diabetes 5 (6.7) 14 (14.3) 12 (20.0) 38 (36.5)  ALT, IU/L 15 (12–19) 15 (12–19) 15 (10–18) 16 (13–20) 0.69  AST, IU/L 18 (16–23) 18 (16–22) 17 (15–20) 18 (15–22) 0.34  ALT/AST ratio 0.86 ± 0.26 0.87 ± 0.28 0.90 ± 0.33 0.88 ± 0.22 0.80  GGT, IU/L 12 (9–17) 12 (10–17) 13 (10–18) 13 (11–22) 0.21  Fetuin-A, g/L 0.6 (0.5–0.7) 0.6 (0.5–0.8) 0.6 (0.5–0.8) 0.6 (0.5–0.7) 0.24 Changes from 1 to 3 y  Change in ALT 1.3 ± 11.7 4.4 ± 6.2 2.4 ± 10.7 3.8 ± 9.3 0.23  Change in AST −1.0 ± 8.6 −0.9 ± 5.5 −3.9 ± 8.1 −1.5 ± 7.5 0.11  Change in ALT/AST ratio 0.14 ± 0.36 0.26 ± 0.25 0.28 ± 0.32 0.25 ± 0.28 0.05  Change in GGT 0.8 ± 9.8 2.3 ± 7.1 4.6 ± 17.6 1.5 ± 12.6 0.38  Change in fetuin-A 0.1 ± 0.2 0.1 ± 0.3 0.1 ± 0.4 0.1 ± 0.3 0.98 P values are for overall comparison across groups by one-way analysis of variance or Kruskal-Wallis test for continuous variables, or either χ2 or Fisher exact test for categorical variables. Continuous variables are presented as mean ± SD (if normally distributed) or median with interquartile range (if skewed). View Large Next, in Table 2, Spearman partial correlation analyses were conducted to assess the relationships between baseline-adjusted changes in each of the hepatic markers from 1 to 3 years with concurrent baseline-adjusted changes in metabolic factors (anthropometrics, insulin sensitivity/resistance, β-cell function, glycemia). We then proceeded to multiple linear regression analyses to determine whether changes in hepatic markers from 1 to 3 years were independently associated with the Matsuda index at 3 years (Table 3) and IGI/HOMA-IR at 3 years (Table 4) as per our main hypothesis, after complete adjustment for covariates. For each of these outcomes, we constructed the following five models to test the hepatic markers in turn: ALT (model I), AST (model II), ALT/AST ratio (model III), GGT (model IV), and fetuin-A (model V). Each regression model was constructed with the following covariates: (1) clinical risk factors for diabetes [age, ethnicity, family history of diabetes, body mass index (BMI) at 1 year, change in BMI from 1 to 3 years, and duration of breastfeeding], (2) the baseline measure of the outcome variable at 1 year, and (3) the indicated hepatic marker at 1 year and its change from 1 to 3 years. Multiple linear regression analyses were similarly conducted for the secondary measures of insulin sensitivity (HOMA-IR) and β-cell function (ISSI-2) in Supplemental Tables 1 and 2, respectively. We then performed multiple linear regression analyses to determine whether changes in hepatic markers from 1 to 3 years were independently associated with fasting glucose at 3 years (Table 5), with model construction performed in the same way. Table 2. Partial Spearman Correlations of Baseline-Adjusted Changes in Hepatic Markers (ALT, AST, ALT/AST Ratio, GGT, Fetuin-A) With Baseline-Adjusted Changes in Metabolic Factors From 1 to 3 Years Postpartum Baseline-Adjusted Changes Between 1 and 3 y Baseline-Adjusted Change in ALT Between 1 and 3 y Baseline-Adjusted Change in AST Between 1 and 3 y Baseline-Adjusted Change in ALT/AST Ratio Between 1 and 3 y Baseline-Adjusted Change in GGT Between 1 and 3 y Baseline-Adjusted Change in Fetuin-A Between 1 and 3 y r P r P r P r P r P BMI 0.10 0.10 0.02 0.78 0.11 0.06 0.22 0.0003 0.06 0.33 Waist 0.15 0.01 0.05 0.42 0.17 0.006 0.13 0.03 0.04 0.53 Matsuda index −0.06 0.31 0.16 0.007 −0.20 0.001 −0.29 <0.0001 −0.24 <0.0001 HOMA-IR 0.07 0.22 −0.12 0.04 0.21 0.0005 0.20 0.0009 0.20 0.0005 Insulinogenic index/HOMA-IR −0.03 0.69 0.04 0.55 −0.08 0.23 −0.06 0.31 −0.11 0.07 ISSI-2 −0.10 0.08 −0.03 0.58 −0.10 0.10 −0.07 0.24 0.01 0.85 Fasting glucose 0.08 0.19 −0.01 0.82 0.09 0.15 0.12 0.04 0.06 0.30 Baseline-Adjusted Changes Between 1 and 3 y Baseline-Adjusted Change in ALT Between 1 and 3 y Baseline-Adjusted Change in AST Between 1 and 3 y Baseline-Adjusted Change in ALT/AST Ratio Between 1 and 3 y Baseline-Adjusted Change in GGT Between 1 and 3 y Baseline-Adjusted Change in Fetuin-A Between 1 and 3 y r P r P r P r P r P BMI 0.10 0.10 0.02 0.78 0.11 0.06 0.22 0.0003 0.06 0.33 Waist 0.15 0.01 0.05 0.42 0.17 0.006 0.13 0.03 0.04 0.53 Matsuda index −0.06 0.31 0.16 0.007 −0.20 0.001 −0.29 <0.0001 −0.24 <0.0001 HOMA-IR 0.07 0.22 −0.12 0.04 0.21 0.0005 0.20 0.0009 0.20 0.0005 Insulinogenic index/HOMA-IR −0.03 0.69 0.04 0.55 −0.08 0.23 −0.06 0.31 −0.11 0.07 ISSI-2 −0.10 0.08 −0.03 0.58 −0.10 0.10 −0.07 0.24 0.01 0.85 Fasting glucose 0.08 0.19 −0.01 0.82 0.09 0.15 0.12 0.04 0.06 0.30 Bold indicates P < 0.05. View Large Table 2. Partial Spearman Correlations of Baseline-Adjusted Changes in Hepatic Markers (ALT, AST, ALT/AST Ratio, GGT, Fetuin-A) With Baseline-Adjusted Changes in Metabolic Factors From 1 to 3 Years Postpartum Baseline-Adjusted Changes Between 1 and 3 y Baseline-Adjusted Change in ALT Between 1 and 3 y Baseline-Adjusted Change in AST Between 1 and 3 y Baseline-Adjusted Change in ALT/AST Ratio Between 1 and 3 y Baseline-Adjusted Change in GGT Between 1 and 3 y Baseline-Adjusted Change in Fetuin-A Between 1 and 3 y r P r P r P r P r P BMI 0.10 0.10 0.02 0.78 0.11 0.06 0.22 0.0003 0.06 0.33 Waist 0.15 0.01 0.05 0.42 0.17 0.006 0.13 0.03 0.04 0.53 Matsuda index −0.06 0.31 0.16 0.007 −0.20 0.001 −0.29 <0.0001 −0.24 <0.0001 HOMA-IR 0.07 0.22 −0.12 0.04 0.21 0.0005 0.20 0.0009 0.20 0.0005 Insulinogenic index/HOMA-IR −0.03 0.69 0.04 0.55 −0.08 0.23 −0.06 0.31 −0.11 0.07 ISSI-2 −0.10 0.08 −0.03 0.58 −0.10 0.10 −0.07 0.24 0.01 0.85 Fasting glucose 0.08 0.19 −0.01 0.82 0.09 0.15 0.12 0.04 0.06 0.30 Baseline-Adjusted Changes Between 1 and 3 y Baseline-Adjusted Change in ALT Between 1 and 3 y Baseline-Adjusted Change in AST Between 1 and 3 y Baseline-Adjusted Change in ALT/AST Ratio Between 1 and 3 y Baseline-Adjusted Change in GGT Between 1 and 3 y Baseline-Adjusted Change in Fetuin-A Between 1 and 3 y r P r P r P r P r P BMI 0.10 0.10 0.02 0.78 0.11 0.06 0.22 0.0003 0.06 0.33 Waist 0.15 0.01 0.05 0.42 0.17 0.006 0.13 0.03 0.04 0.53 Matsuda index −0.06 0.31 0.16 0.007 −0.20 0.001 −0.29 <0.0001 −0.24 <0.0001 HOMA-IR 0.07 0.22 −0.12 0.04 0.21 0.0005 0.20 0.0009 0.20 0.0005 Insulinogenic index/HOMA-IR −0.03 0.69 0.04 0.55 −0.08 0.23 −0.06 0.31 −0.11 0.07 ISSI-2 −0.10 0.08 −0.03 0.58 −0.10 0.10 −0.07 0.24 0.01 0.85 Fasting glucose 0.08 0.19 −0.01 0.82 0.09 0.15 0.12 0.04 0.06 0.30 Bold indicates P < 0.05. View Large Table 3. Hepatic Markers at 1 Year and Their Changes From 1 to 3 Years as Predictors of the Matsuda Index at 3 Years Postpartum Model/Hepatic Markers β t P I  ALT at 1 y −0.003 −0.78 0.44  Change in ALT from 1 to 3 y −0.01 −2.61 0.01 II  AST at 1 y 0.002 0.28 0.78  Change in AST from 1 to 3 y 0.02 3.23 0.001 III  ALT/AST ratio at 1 y −0.24 −1.99 0.05  Change in ALT/AST ratio from 1 to 3 y −0.59 −5.47 <0.0001 IV  GGT at 1 y −0.004 −1.42 0.16  Change in GGT from 1 to 3 y −0.008 −3.20 0.002 V  Fetuin-A at 1 y −0.47 −3.17 0.002  Change in fetuin-A from 1 to 3 y −0.40 −3.56 0.0004 Model/Hepatic Markers β t P I  ALT at 1 y −0.003 −0.78 0.44  Change in ALT from 1 to 3 y −0.01 −2.61 0.01 II  AST at 1 y 0.002 0.28 0.78  Change in AST from 1 to 3 y 0.02 3.23 0.001 III  ALT/AST ratio at 1 y −0.24 −1.99 0.05  Change in ALT/AST ratio from 1 to 3 y −0.59 −5.47 <0.0001 IV  GGT at 1 y −0.004 −1.42 0.16  Change in GGT from 1 to 3 y −0.008 −3.20 0.002 V  Fetuin-A at 1 y −0.47 −3.17 0.002  Change in fetuin-A from 1 to 3 y −0.40 −3.56 0.0004 Five multiple linear regression models were constructed to evaluate the following hepatic markers at 1 y and their changes from 1 to 3 y: ALT (model I), AST (model II), ALT/AST ratio (model III), GGT (model IV), and fetuin-A (model V). Each model was adjusted for age, ethnicity, family history of T2DM, BMI at 1 y, change in BMI from 1 to 3 y, breastfeeding, and the Matsuda index at 1 y. View Large Table 3. Hepatic Markers at 1 Year and Their Changes From 1 to 3 Years as Predictors of the Matsuda Index at 3 Years Postpartum Model/Hepatic Markers β t P I  ALT at 1 y −0.003 −0.78 0.44  Change in ALT from 1 to 3 y −0.01 −2.61 0.01 II  AST at 1 y 0.002 0.28 0.78  Change in AST from 1 to 3 y 0.02 3.23 0.001 III  ALT/AST ratio at 1 y −0.24 −1.99 0.05  Change in ALT/AST ratio from 1 to 3 y −0.59 −5.47 <0.0001 IV  GGT at 1 y −0.004 −1.42 0.16  Change in GGT from 1 to 3 y −0.008 −3.20 0.002 V  Fetuin-A at 1 y −0.47 −3.17 0.002  Change in fetuin-A from 1 to 3 y −0.40 −3.56 0.0004 Model/Hepatic Markers β t P I  ALT at 1 y −0.003 −0.78 0.44  Change in ALT from 1 to 3 y −0.01 −2.61 0.01 II  AST at 1 y 0.002 0.28 0.78  Change in AST from 1 to 3 y 0.02 3.23 0.001 III  ALT/AST ratio at 1 y −0.24 −1.99 0.05  Change in ALT/AST ratio from 1 to 3 y −0.59 −5.47 <0.0001 IV  GGT at 1 y −0.004 −1.42 0.16  Change in GGT from 1 to 3 y −0.008 −3.20 0.002 V  Fetuin-A at 1 y −0.47 −3.17 0.002  Change in fetuin-A from 1 to 3 y −0.40 −3.56 0.0004 Five multiple linear regression models were constructed to evaluate the following hepatic markers at 1 y and their changes from 1 to 3 y: ALT (model I), AST (model II), ALT/AST ratio (model III), GGT (model IV), and fetuin-A (model V). Each model was adjusted for age, ethnicity, family history of T2DM, BMI at 1 y, change in BMI from 1 to 3 y, breastfeeding, and the Matsuda index at 1 y. View Large Table 4. Hepatic Markers at 1 Year and Their Changes From 1 to 3 Years as Predictors of IGI/HOMA-IR at 3 Years Postpartum Model/Hepatic Markers β t P I  ALT at 1 y −0.003 −0.31 0.75  Change in ALT from 1 to 3 y −0.01 −1.78 0.08 II  AST at 1 y 0.02 1.90 0.06  Change in AST from 1 to 3 y 0.01 1.52 0.13 III  ALT/AST ratio at 1 y −0.59 −2.45 0.01  Change in ALT/AST ratio from 1 to 3 y −0.73 −3.35 0.001 IV  GGT at 1 y 0.0006 0.12 0.90  Change in GGT from 1 to 3 y −0.002 −0.39 0.70 V  Fetuin-A at 1 y −0.26 −0.85 0.40  Change in fetuin-A from 1 to 3 y −0.25 −1.08 0.28 Model/Hepatic Markers β t P I  ALT at 1 y −0.003 −0.31 0.75  Change in ALT from 1 to 3 y −0.01 −1.78 0.08 II  AST at 1 y 0.02 1.90 0.06  Change in AST from 1 to 3 y 0.01 1.52 0.13 III  ALT/AST ratio at 1 y −0.59 −2.45 0.01  Change in ALT/AST ratio from 1 to 3 y −0.73 −3.35 0.001 IV  GGT at 1 y 0.0006 0.12 0.90  Change in GGT from 1 to 3 y −0.002 −0.39 0.70 V  Fetuin-A at 1 y −0.26 −0.85 0.40  Change in fetuin-A from 1 to 3 y −0.25 −1.08 0.28 Five multiple linear regression models were constructed to evaluate the following hepatic markers at 1 y and their changes from 1 to 3 y: ALT (model I), AST (model II), ALT/AST ratio (model III), GGT (model IV), and fetuin-A (model V). Each model was adjusted for age, ethnicity, family history of T2DM, BMI at 1 y, change in BMI from 1 to 3 y, breastfeeding, and IGI/HOMA-IR at 1 y. View Large Table 4. Hepatic Markers at 1 Year and Their Changes From 1 to 3 Years as Predictors of IGI/HOMA-IR at 3 Years Postpartum Model/Hepatic Markers β t P I  ALT at 1 y −0.003 −0.31 0.75  Change in ALT from 1 to 3 y −0.01 −1.78 0.08 II  AST at 1 y 0.02 1.90 0.06  Change in AST from 1 to 3 y 0.01 1.52 0.13 III  ALT/AST ratio at 1 y −0.59 −2.45 0.01  Change in ALT/AST ratio from 1 to 3 y −0.73 −3.35 0.001 IV  GGT at 1 y 0.0006 0.12 0.90  Change in GGT from 1 to 3 y −0.002 −0.39 0.70 V  Fetuin-A at 1 y −0.26 −0.85 0.40  Change in fetuin-A from 1 to 3 y −0.25 −1.08 0.28 Model/Hepatic Markers β t P I  ALT at 1 y −0.003 −0.31 0.75  Change in ALT from 1 to 3 y −0.01 −1.78 0.08 II  AST at 1 y 0.02 1.90 0.06  Change in AST from 1 to 3 y 0.01 1.52 0.13 III  ALT/AST ratio at 1 y −0.59 −2.45 0.01  Change in ALT/AST ratio from 1 to 3 y −0.73 −3.35 0.001 IV  GGT at 1 y 0.0006 0.12 0.90  Change in GGT from 1 to 3 y −0.002 −0.39 0.70 V  Fetuin-A at 1 y −0.26 −0.85 0.40  Change in fetuin-A from 1 to 3 y −0.25 −1.08 0.28 Five multiple linear regression models were constructed to evaluate the following hepatic markers at 1 y and their changes from 1 to 3 y: ALT (model I), AST (model II), ALT/AST ratio (model III), GGT (model IV), and fetuin-A (model V). Each model was adjusted for age, ethnicity, family history of T2DM, BMI at 1 y, change in BMI from 1 to 3 y, breastfeeding, and IGI/HOMA-IR at 1 y. View Large Table 5. Hepatic Markers at 1 Year and Their Changes From 1 to 3 Years as Predictors of Fasting Glucose at 3 Years Postpartum Model/Hepatic Markers β t P I  ALT at 1 y 0.004 1.01 0.31  Change in ALT from 1 to 3 y 0.007 1.98 0.05 II  AST at 1 y −0.003 −0.55 0.59  Change in AST from 1 to 3 y −0.002 −0.45 0.65 III  ALT/AST ratio at 1 y 0.20 1.62 0.11  Change in ALT/AST ratio from 1 to 3 y 0.29 2.55 0.01 IV  GGT at 1 y −0.003 −1.17 0.24  Change in GGT from 1 to 3 y 0.0008 0.32 0.75 V  Fetuin-A at 1 y −0.08 −0.53 0.59  Change in fetuin-A from 1 to 3 y 0.01 0.10 0.92 Model/Hepatic Markers β t P I  ALT at 1 y 0.004 1.01 0.31  Change in ALT from 1 to 3 y 0.007 1.98 0.05 II  AST at 1 y −0.003 −0.55 0.59  Change in AST from 1 to 3 y −0.002 −0.45 0.65 III  ALT/AST ratio at 1 y 0.20 1.62 0.11  Change in ALT/AST ratio from 1 to 3 y 0.29 2.55 0.01 IV  GGT at 1 y −0.003 −1.17 0.24  Change in GGT from 1 to 3 y 0.0008 0.32 0.75 V  Fetuin-A at 1 y −0.08 −0.53 0.59  Change in fetuin-A from 1 to 3 y 0.01 0.10 0.92 Five multiple linear regression models were constructed to evaluate the following hepatic markers at 1 y and their changes from 1 to 3 y: ALT (model I), AST (model II), ALT/AST ratio (model III), GGT (model IV), and fetuin-A (model V). Each model was adjusted for age, ethnicity, family history of T2DM, BMI at 1 y, change in BMI from 1 to 3 y, breastfeeding, and fasting glucose at 1 y. View Large Table 5. Hepatic Markers at 1 Year and Their Changes From 1 to 3 Years as Predictors of Fasting Glucose at 3 Years Postpartum Model/Hepatic Markers β t P I  ALT at 1 y 0.004 1.01 0.31  Change in ALT from 1 to 3 y 0.007 1.98 0.05 II  AST at 1 y −0.003 −0.55 0.59  Change in AST from 1 to 3 y −0.002 −0.45 0.65 III  ALT/AST ratio at 1 y 0.20 1.62 0.11  Change in ALT/AST ratio from 1 to 3 y 0.29 2.55 0.01 IV  GGT at 1 y −0.003 −1.17 0.24  Change in GGT from 1 to 3 y 0.0008 0.32 0.75 V  Fetuin-A at 1 y −0.08 −0.53 0.59  Change in fetuin-A from 1 to 3 y 0.01 0.10 0.92 Model/Hepatic Markers β t P I  ALT at 1 y 0.004 1.01 0.31  Change in ALT from 1 to 3 y 0.007 1.98 0.05 II  AST at 1 y −0.003 −0.55 0.59  Change in AST from 1 to 3 y −0.002 −0.45 0.65 III  ALT/AST ratio at 1 y 0.20 1.62 0.11  Change in ALT/AST ratio from 1 to 3 y 0.29 2.55 0.01 IV  GGT at 1 y −0.003 −1.17 0.24  Change in GGT from 1 to 3 y 0.0008 0.32 0.75 V  Fetuin-A at 1 y −0.08 −0.53 0.59  Change in fetuin-A from 1 to 3 y 0.01 0.10 0.92 Five multiple linear regression models were constructed to evaluate the following hepatic markers at 1 y and their changes from 1 to 3 y: ALT (model I), AST (model II), ALT/AST ratio (model III), GGT (model IV), and fetuin-A (model V). Each model was adjusted for age, ethnicity, family history of T2DM, BMI at 1 y, change in BMI from 1 to 3 y, breastfeeding, and fasting glucose at 1 y. View Large Finally, we conducted forward selection logistic regression analyses to determine independent predictors of prediabetes/diabetes at 3 years [defined as IFG, IGT, combined IFG/IGT, or diabetes, as per CDA guidelines (13)]. The covariates for selection were as follows: (1) diabetes risk factors (age, ethnicity, family history of diabetes, BMI at 1 year, change in BMI from 1 to 3 years, duration of breastfeeding), (2) glucose tolerance status at 1 year, and (3) all of the hepatic markers at 1 year (ALT, AST, GGT, fetuin-A) and the change in each hepatic marker from 1 to 3 years (Fig. 1). Age, ethnicity, family history of diabetes, BMI at 1 year, and duration of breastfeeding were forced into the model as major clinical risk factors for diabetes. The forward selection model was repeated with the inclusion of ALT/AST ratio and its change, in place of ALT and AST and their changes. Figure 1. View largeDownload slide Forward selection logistic regression model of (outcome) prediabetes/diabetes at 3 y postpartum, with the following covariates available for selection: age, ethnicity, family history of diabetes, BMI at 1 y, change in BMI from 1 to 3 y, duration of breastfeeding, glucose tolerance status at 1 y, all of the hepatic markers at 1 y (ALT, AST, GGT, and fetuin-A), and change in each hepatic marker from 1 to 3 y. Major clinical risk factors for diabetes (age, ethnicity, family history of diabetes, BMI at 1 y, and duration of breastfeeding) were forced into the model. Figure 1. View largeDownload slide Forward selection logistic regression model of (outcome) prediabetes/diabetes at 3 y postpartum, with the following covariates available for selection: age, ethnicity, family history of diabetes, BMI at 1 y, change in BMI from 1 to 3 y, duration of breastfeeding, glucose tolerance status at 1 y, all of the hepatic markers at 1 y (ALT, AST, GGT, and fetuin-A), and change in each hepatic marker from 1 to 3 y. Major clinical risk factors for diabetes (age, ethnicity, family history of diabetes, BMI at 1 y, and duration of breastfeeding) were forced into the model. Results Table 1 shows the characteristics of the study population at 1 year and 3 years postpartum, stratified into the following four groups based on their preceding gestational glucose tolerance status: (1) normal GCT NGT (n = 75), (2) abnormal GCT NGT (n = 98), (3) GIGT (n = 59), and (4) GDM (n = 104). At 1 year postpartum, the groups did not differ in age, ethnicity, family history of diabetes, duration of breastfeeding, smoking status, or BMI. As expected, at 1 year postpartum, there was a progressive decrease in insulin sensitivity (Matsuda index: P = 0.003) and β-cell function (IGI/HOMA-IR: P < 0.0001) from normal GCT NGT to abnormal GCT NGT to GIGT to GDM. Accordingly, there was a concomitant progressive rise in fasting glucose and 2-hour glucose across the four groups (both P < 0.0001), coupled with a stepwise increase in the prevalence of dysglycemia at 1 year from 2.8% to 12.8% to 22.2% to 29.6% (P < 0.0001), most of which (95%) was prediabetes. Of note, the hepatic markers ALT, AST, ALT/AST ratio, GGT, and fetuin-A did not differ across groups. At 3 years postpartum, the same patterns were apparent across the four gestational glucose tolerance groups for insulin sensitivity, β-cell function, fasting glucose, 2-hour glucose, and current glucose tolerance status. As before, the hepatic markers did not differ among the groups at 3 years. However, the change in ALT/AST ratio from 1 to 3 years differed across the groups (P = 0.05), with normal GCT NGT showing a smaller change than the three gestational dysglycemia groups. Changes in the other hepatic markers did not differ across the groups. After adjustment for diabetes risk factors (age, ethnicity, family history of diabetes, BMI, duration of breastfeeding) and current glucose tolerance, there were no statistically significant differences between the four gestational glucose tolerance groups in mean adjusted levels of any of the hepatic markers at 3 years (data not shown). Thus, between 1 and 3 years postpartum, the interval change in ALT/AST ratio was the only apparent hepatic marker difference between these four groups that reflected different degrees of future diabetic risk. Changes in hepatic markers and metabolic outcomes over time We next performed Spearman partial correlation analyses to evaluate the relationships between baseline-adjusted changes in hepatic markers and baseline-adjusted changes in metabolic factors from 1 to 3 years postpartum (Table 2). These analyses revealed that baseline-adjusted changes in ALT/AST ratio, GGT, and fetuin-A were inversely associated with baseline-adjusted changes in the Matsuda index, suggesting that increasing concentrations of these hepatic markers over time were accompanied by worsening insulin sensitivity. We next performed multiple linear regression analyses to determine whether any of the hepatic markers or their changes over time were independent predictors of the Matsuda index at 3 years, after adjustment for diabetes risk factors (age, ethnicity, family history of T2DM, breastfeeding, BMI at 1 year, change in BMI from 1 to 3 years) and the Matsuda index at 1 year (Table 3). Importantly, for each hepatic marker, its respective change from 1 to 3 years was an independent predictor of insulin sensitivity (Matsuda) at 3 years (Table 3). These relationships were strongest for ALT/AST ratio (t = −5.47, P < 0.0001) and fetuin-A (t = −3.56, P < 0.0001), both of which also showed independent associations of their baseline measures at 1 year with lower insulin sensitivity at 3 years. Similarly, the baseline measures at 1 year and changes from 1 to 3 years in ALT/AST ratio and fetuin-A both emerged as independent predictors of HOMA-IR at 3 years (Supplemental Table 1). Using the same modeling approach, we performed similar multiple linear regression analyses of our primary and secondary measures of β-cell function in Table 4 and Supplemental Table 2, respectively. These analyses showed that an increase in ALT/AST ratio from 1 to 3 years predicted poorer β-cell function as measured by either IGI/HOMA-IR (t = −3.35, P = 0.001) (Table 4) or ISSI-2 (t = −2.33, P = 0.02) (Supplemental Table 2). Moreover, similar multiple linear regression analyses of fasting glucose revealed that the increase in ALT/AST ratio from 1 to 3 years predicted higher fasting glycemia at 3 years (t = 2.55, P = 0.01) (Table 5). To evaluate the robustness of the findings from these multiple linear regression models in Tables 3 to 5 and Supplemental Tables 1 to 2, we performed a series of sensitivity analyses. The findings were largely unchanged in sensitivity analyses in which waist circumference at 1 year and the change in waist circumference from 1 to 3 years were included in the models, in place of baseline BMI and its change, respectively (data not shown). To address the possibility of false discovery, we also applied the correction method of Storey and confirmed that the findings remained unchanged (data not shown). Thus, from all of these analyses, ALT/AST ratio and fetuin-A emerged as hepatic markers that predicted pathophysiologic determinants of diabetes. Hepatic markers and prediabetes/diabetes Finally, we performed logistic regression analyses to determine whether any of the hepatic markers or their changes over time were independent predictors of prediabetes/diabetes at 3 years (Fig. 1). In a forward selection analysis that included baseline ALT, AST, GGT, and fetuin-A and their respective changes from 1 to 3 years as potential covariates, fetuin-A at 1 year emerged as a significant predictor of prediabetes/diabetes at 3 years (OR, 1.38; 95% CI, 1.01 to 1.88), accompanied by glucose intolerance at 1 year (OR, 11.0; 95% CI, 5.03 to 24.14), BMI at 1 year (OR, 1.44; 95% CI, 1.00 to 2.06), and the change in BMI from 1 to 3 years (OR, 1.55; 95% CI, 1.09 to 2.22). Furthermore, on sensitivity analyses with inclusion of ALT/AST ratio in place of ALT and AST, these findings were unchanged, with fetuin-A at 1 year again predicting prediabetes/diabetes (OR, 1.38; 95% CI, 1.01 to 1.88) (data not shown). Discussion In this study, we demonstrate three main findings. First, at both 1 and 3 years postpartum, the hepatic markers ALT, AST, GGT, and fetuin-A did not differ across four recent gestational tolerance groups reflecting different degrees of future diabetic risk. However, the intervening change in the ALT/AST ratio did differ between the groups. Second, changes over time in hepatic markers, particularly the ALT/AST ratio and fetuin-A, tracked with changes in insulin sensitivity and β-cell function. Third, and most important, at this early point in the natural history of T2DM, the ALT/AST ratio and fetuin-A emerged as independent predictors of fasting glycemia and prediabetes/diabetes, respectively. Taken together, these findings support a pathophysiologic basis in the relationship between hepatic markers and subsequent risk of T2DM. Although previous reports have linked fatty liver with insulin resistance in women with a history of GDM (19–21), few studies have evaluated circulating hepatic markers and their implications for diabetic risk in this population. In a cross-sectional study at mean 8 to 9 months postpartum, Rottenkolber et al. (22) reported that circulating fetuin-A was higher in 96 women with recent GDM compared with 51 controls. Forbes et al. (20) found that ALT and GGT did not differ between 110 women with previous GDM and 113 without such a history, at a mean 6 to 7 years postpartum. However, limitations of the studies to date have included modest samples sizes, cross-sectional evaluation at a single point in time, and potential heterogeneity in comparators (i.e., categorized as non-GDM, with identification after delivery). In this context, strengths of the current study are the prospective ascertainment of gestational glucose tolerance, yielding a well-characterized cohort of 336 women across the full spectrum of gestational glucose tolerance (from normal to GDM), coupled with assessment of both hepatic markers and metabolic outcomes on two occasions 2 years apart. With this approach, we demonstrate that, at both 1 and 3 years postpartum, ALT, AST, GGT, and fetuin-A did not differ across four recent gestational glucose tolerance groups that reflect distinct degrees of future diabetic risk. This study design also made it possible to evaluate the changes in hepatic markers over 2 years in these risk groups. In this regard, we observed that the three groups with higher future diabetic risk (GDM, GIGT, abnormal GCT NGT) experienced a greater increase in the ALT/AST ratio between 1 and 3 years than did the group with the lowest such risk (normal GCT NGT). Although an increased AST/ALT ratio (De Ritis ratio) has long been discussed clinically as a marker of liver disease (23), there has been limited previous evaluation in relation to the risk of T2DM. In a recent cross-sectional analysis from the Korean National Health and Nutrition Examination Survey, Ko et al. (24) reported that a lower AST/ALT ratio within the physiological range (i.e., hence higher ALT/AST ratio) was associated with a greater likelihood of IFG and T2DM. Similarly, in a cross-sectional study of nonobese Japanese adults, the ALT/AST ratio was associated with insulin resistance, as measured by HOMA-IR (25). Interestingly, when the Insulin Resistance and Atherosclerosis Study reported that this ratio predicted metabolic syndrome, the only component disorder thereof with which it was significantly associated in a series of separate models was IFG (26). Against this background, our findings suggest that, in this study population that is very early in the natural history of T2DM, the ALT/AST ratio may amplify a signal of future diabetic risk that is not yet detectable with individual liver enzymes alone. In this context, our evaluation of hepatic makers and metabolic function at two points in time is particularly informative by enabling not only the assessment of cross-sectional and longitudinal associations between these exposures and outcomes but also the relationship between their respective changes over time. We show that, although changes in all of the liver markers related to changes in insulin sensitivity (Table 3), the strongest predictors thereof were ALT/AST ratio and fetuin-A. Moreover, an increase in the ALT/AST ratio was the only hepatic marker that independently predicted lower β-cell function at 3 years, and it did so for both β-cell measures (Table 4, Supplemental Table 2). Finally, consistent with its associations with insulin resistance and β-cell dysfunction, rising ALT/AST ratio emerged as an independent predictor of higher fasting glucose at this early point in the natural history of diabetes (Table 5). Few previous studies have evaluated serial measurements of liver enzymes in relation to diabetic risk, although a secondary case-control analysis of the West of Scotland Coronary Prevention Study found elevations in ALT at 18 months, 12 months, and 6 months prior to the diagnosis of diabetes in 86 middle-aged male participants (27). The current study extends this concept by showing that a rising ALT/AST ratio tracks with insulin resistance, β-cell dysfunction, and fasting glucose early in the natural history of diabetes in young women. Although it has generally been believed that hepatic fat underlies the association between liver enzymes and diabetic risk (8, 9), a growing body of evidence suggests that this mechanistic basis may not fully apply to fetuin-A. First, in vitro studies have shown that fetuin-A reversibly binds the insulin receptor tyrosine kinase in peripheral tissues and can thereby directly induce insulin resistance (28, 29). Second, the Multiethnic Study of Atherosclerosis recently reported that baseline fetuin-A independently predicted incident diabetes in women, after adjustment for liver fat content (measured by computed tomography) (7). Third, in the Nurses’ Health Study, the circulating fetuin-A concentration predicted the subsequent risk of T2DM even after adjustment for liver enzymes (ALT and GGT) (6). Our findings are consistent with these observations and extend this literature in two ways. First, we demonstrate that both baseline fetuin-A and its change from 1 to 3 years postpartum were independently associated with lower whole-body insulin sensitivity (Table 3). Second, fetuin-A at 1 year emerged as an independent predictor of prediabetes/diabetes at 3 years in a forward selection analysis that included all of the liver enzymes, both at baseline and their change over time. Of note, it did so in the absence of an independent association with fasting glycemia, consistent with previous observations suggesting that elevated fetuin-A is a feature of IGT but not IFG (30, 31). Overall, our findings link both ALT/AST ratio and fetuin-A to early dysglycemia in young women but support the concept that they have distinct mechanistic bases in this regard. A limitation of this study is that some factors that could affect liver enzymes have not been evaluated. These include maternal infections, diet, and lifestyle practices. However, the current analyses were adjusted for major diabetes risk factors (age, ethnicity, family history, BMI) and breastfeeding history [which has been shown to have sustained effects on insulin sensitivity and long-term diabetic risk (32)]. Another limitation is that insulin sensitivity and β-cell function were assessed with OGTT-based surrogate indices rather than clamp studies. However, their time-consuming and invasive nature would have made clamp studies difficult to complete on two occasions over 2 years in 336 new mothers. Moreover, the Matsuda index, HOMA-IR, ISSI-2, and IGI/HOMA-IR are validated measures that have been widely used in previous studies (10–12, 14–18), and the serial OGTTs on which they were determined made it possible to also assess glucose tolerance status. In conclusion, at both 1 and 3 years postpartum, ALT, AST, GGT, and fetuin-A did not differ across four recent gestational tolerance groups reflecting different degrees of future diabetic risk. However, the interval change in the ALT/AST ratio differed between these groups. Moreover, change in the ALT/AST ratio and fetuin-A tracked with changes in insulin sensitivity and β-cell function, thereby linking these hepatic markers to the pathologic determinants of T2DM. Most important, the ALT/AST ratio and fetuin-A emerged as independent predictors of fasting glycemia and prediabetes/diabetes, respectively. Taken together, these findings thus support a pathophysiologic basis in the relationship between changes in circulating hepatic markers and diabetic risk very early in the natural history of T2DM in women. Abbreviations: Abbreviations: ALT alanine aminotransferase AST aspartate aminotransferase BMI body mass index CDA Canadian Diabetes Association GCT glucose challenge test GDM gestational diabetes mellitus GGT γ-glutamyltransferase GIGT gestational impaired glucose tolerance HOMA-IR homeostasis model assessment of insulin resistance IFG impaired fasting glucose IGI insulinogenic index IGT impaired glucose tolerance ISSI-2 Insulin Secretion-Sensitivity Index–2 NDDG National Diabetes Data Group NGT normal glucose tolerance OGTT oral glucose tolerance test T2DM type 2 diabetes mellitus Acknowledgments Financial Support: This study was supported by operating grants from the Canadian Institutes of Health Research (CIHR)(MOP-84206) and Canadian Diabetes Association (CDA)(CDA-OG-3-15-4924-RR) to R.R. A.J.H. holds a Tier-II Canada Research Chair in Diabetes Epidemiology. B.Z. holds the Sam and Judy Pencer Family Chair in Diabetes Research at Mount Sinai Hospital and University of Toronto. R.R. is supported by a Heart and Stroke Foundation of Ontario Mid-Career Investigator Award and holds the Boehringer Ingelheim Chair in Beta-cell Preservation, Function and Regeneration at Mount Sinai Hospital. Author Contributions: R.R., A.J.H., P.W.C., M.S., and B.Z. designed and implemented the study. R.R. and C.Y. contributed to the analysis plan and interpretation of the data. C.Y. performed the statistical analyses. L.P. wrote the first draft. All authors critically revised the manuscript for important intellectual content. All authors approved the final manuscript. R.R. is guarantor, had full access to all of the data in the study, and takes responsibility for the integrity of the data and the accuracy of the data analysis. Disclosure Summary: The authors have nothing to disclose. References 1. Hanley AJ , Williams K , Festa A , Wagenknecht LE , D’Agostino RB Jr , Kempf J , Zinman B , Haffner SM ; Insulin Resistance Atherosclerosis Study . 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Journal of Clinical Endocrinology and MetabolismOxford University Press

Published: Apr 20, 2018

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