Journal Logo

CLINICAL TRANSPLANTATION

Assessment of insulin sensitivity based on a fasting blood sample in men with liver cirrhosis before and after liver transplantation1

Perseghin, Gianluca2 3 5; Caumo, Andrea2; Mazzaferro, Vincenzo4; Pulvirenti, Andrea4; Piceni Sereni, Lucia2; Romito, Raffaele4; Lattuada, Guido2; Coppa, Jorgelina4; Costantino, Federica2; Regalia, Enrico4; Luzi, Livio2 3

Author Information
doi: 10.1097/01.TP.0000079252.94857.8D
  • Free

Abstract

Glucose intolerance and diabetes are highly prevalent among patients with chronic liver diseases (1,2). Insulin resistance constitutes a primary pathogenic event, and patients with additional β-cell secretory defects develop overt diabetes (3,4). The insulin resistance syndrome associated with liver cirrhosis is also important because it may influence the natural history of the disease (4,5). In addition, nonalcoholic fatty liver disease (with a prevalence of 23.5% in the United States) (6) has been recognized as part of the insulin resistance syndrome (7), implying a pathogenic role for insulin resistance (8). The recognition of insulin resistance, therefore, has clinical and investigational relevance in patients with liver diseases.

The insulin clamp technique (9) is considered the “gold standard” (10) for in vivo quantification of insulin sensitivity; but this method is complex and expensive. A well-accepted alternative is the minimal model analysis of a frequently sampled intravenous (IV) glucose tolerance test (11), which is less laborious but is not as simple as required in large scale studies. Homeostasis model assessment (HOMA) of insulin sensitivity, based on the product of the fasting plasma insulin and glucose concentrations, measured in a single blood sample, divided by a constant (12), was shown to be a reliable surrogate measure of in vivo insulin sensitivity in humans when compared with the clamp (13). Because the distribution of fasting insulin values is skewed, converting the insulin-glucose product to the logarithm and the reciprocal of the insulin-glucose product has been proven advantageous (14). The new index is called Quantitative Insulin sensitivity Check Index (QUICKI). These indices were shown to estimate insulin sensitivity in conditions classically characterized by insulin resistance (type 2 diabetes, obesity, hypertension, cardiovascular atherosclerotic disease) (15). This study was undertaken to ascertain whether these indices may also be used as surrogates of insulin sensitivity in patients with liver cirrhosis and to test whether they may be useful to prospectively monitor insulin sensitivity in patients after liver transplantation (LTx).

EXPERIMENTAL PROCEDURES

Subjects

Twenty patients with postnecrotic cirrhosis (Child-Pugh class A or B) complicated by small unresectable hepatocellular carcinoma (<5-cm single tumor, <3-cm diameter for multiple tumors) were recruited in the Hepato-Pancreatic Surgery and Liver Transplantation Unit of the National Cancer Institute. Twelve patients were also diagnosed with diabetes (three separate fasting plasma glucose >7.8 mmol/L). None had metastatic disease at the time of transplantation or when this study was performed. Thirty-six liver transplant patients were also recruited; 14 of them were also diagnosed with diabetes. All patients were in stable clinical and nutritional condition; liver function was normal with the exception of a slight increase of alanine aminotransferase and aspartate aminotransferase. Twenty-five healthy subjects matched for anthropometric features served as control group. Clinical and laboratory characteristics of study groups are summarized in Table 1. All study groups were predominantly male. Approximately 2 years after LTx, 10 of the 20 patients with postnecrotic cirrhosis were studied again. Their clinical and laboratory characteristics before and after the surgical procedure are summarized in Table 2. Subjects were fully informed of the possible risk of the study and gave their consent. The protocol was approved by the Ethical Committee of the Istituto Scientifico H San Raffaele.

T1-16
Table 1:
Anthropometric, laboratory, and insulin sensitivity characteristics of the study groups
T2-16
Table 2:
Anthropometric, laboratory, and insulin sensitivity characteristics of the patients with cirrhosis studied before and after LTx in a prospective fashion

Experimental Protocol

Subjects were instructed to consume an isocaloric diet and to abstain from exercise activity for 3 weeks before the studies. The use of drugs with potential effects on glucose and insulin metabolism was discontinued for a washout period of 3 days, including oral hypoglycemic agents. At the time of the study, transplant recipients were treated with cyclosporine and in some cases with prednisone. Subjects took their immunosuppressive medications according to a regular schedule, except for prednisone, which was held until completion of the study. Subjects were studied by means of the euglycemic-hyperinsulinemic clamp to assess insulin sensitivity after a 10-hr overnight fast. Patients with diabetes were instructed to receive the last doses of intermediate and short-acting insulin, respectively, 24 and 12 hr before the study. Subjects were admitted to the Metabolic Unit of the Division of Internal Medicine I of the Istituto Scientifico H San Raffaele at 07:00 a.m. after a 10-hr overnight fast.

A Teflon catheter was inserted into the antecubital vein for infusions, and an additional catheter was inserted retrogradely into a wrist vein for blood sampling. The hand was kept in a heated box (50°C) throughout the experiment to allow sampling of arterialized venous blood. Blood samples for postabsorptive plasma glucose and free-insulin were performed in triplicate. Thereafter the insulin clamp was performed as previously described (16). Insulin was infused at 40 mU/[m2·min] to reach a plasma insulin concentration of approximately 450 pmol/L. When fasting hyperglycemia was present, plasma glucose concentration was allowed to drop toward the euglycemic level, and thereafter we attempted to maintain a plasma glucose concentration of 5 mmol/L as in the normoglycemic individuals for 150 min by means of a variable infusion of 20% dextrose. This procedure determined the performance of an isoglycemic rather than a euglycemic clamp in the hyperglycemic individuals, but this was preferable because acute changes in glycemia may affect insulin sensitivity. Blood samples for plasma glucose and free-insulin were drawn every 15 min throughout the study.

Analytical Procedures

Plasma glucose was measured with a Beckman glucose analyzer (16) and coefficients of variation (CV) were 1.7±0.1% and 3.0±0.4% in the fasting and clamp conditions, respectively. Plasma free-insulin was measured by means of an RIA Incstar (Stillwater, MN) as previously described (17). CV was 10.2±1.7% and 6.1±0.7% in the fasting and clamp conditions, respectively.

Calculations

The glucose infusion rate (GIR) was measured in steady-state conditions (CV was 2.3±0.3%) during the 120 to 150 min of the insulin clamp and expressed as mg/[kg·min]. Insulin sensitivity (SIclamp) was calculated as follows: GIR/(ΔI × G), where ΔI was the increment of plasma insulin concentrations during the 120 to 150 min of the clamp with respect to the basal condition, and G was the plasma glucose concentration during the 120 to 150 min of the clamp (18,19). In these hyperinsulinemic conditions, endogenous glucose production is completely suppressed; in nondiabetic patients with liver cirrhosis and in diabetic and nondiabetic liver transplant patients, we previously demonstrated a normal suppression of the endogenous glucose production (3). In the same work, it was shown that cirrhotic patients with diabetes had a minimal but significant impairment of the insulin-dependent suppression of the endogenous glucose production. The lack of measurement of the endogenous glucose production during the clamp procedure might therefore had an impact on the relationship between the SIclamp and HOMA or QUICKI in patients with liver cirrhosis and diabetes. HOMA provided equations for estimating insulin resistance MATHand insulin sensitivity MATHwhere I0 (μU/ml) was the fasting insulin concentration, and G0 (mmol/L) was the fasting glucose concentration, and 22.5 represents a constant applied to correct the value to unity as previously described (12,13). QUICKI was calculated as previously described (14):MATH

Statistical Analysis

All data are presented as a mean ± SD. Steady state was defined as a nonsignificant correlation with time (P >0.05) of the variable using standard linear regression. Relationship between variables was assessed by means of simple correlation (r) analysis. Analysis of variance and chi-square analysis were used for comparison between groups, and Bonferroni adjustment was used for posthoc comparisons. Student t test for paired data was used for comparison between patients with liver cirrhosis studied longitudinally before and after LTx.

RESULTS

Anthropometric and Laboratory Characteristics of the Study Subjects

All study groups were predominantly male (Table 1). Patients with liver cirrhosis and LTx patients were slightly older than normal patients, but posthoc testing did not reach statistical difference. Body mass index was also not different among groups. Fasting plasma glucose was significantly increased in patients with liver cirrhosis and LTx patients (P =0.05) when compared with normal patients. Fasting plasma free-insulin concentration was increased in patients with liver cirrhosis with respect to LTx patients and normal individuals (P <0.01).

Insulin Sensitivity in the General Population

GIR and SIclamp and HOMA-IS and QUICKI were significantly reduced in patients with liver cirrhosis when compared with LTx patients and normal individuals (P <0.01;Table 1). Fasting plasma glucose was associated with GIR (r =−0.45, P <0.0001) and SIclamp (r =−0.45, P <0.0001), and the same was observed for fasting plasma insulin (r =−0.56, P <0.0001 and r =−0.57, P <0.0001, respectively). GIR was associated with HOMA-IS (r =0.68, P <0.0001) and QUICKI (r =0.72, P <0.0001) as well as SIclamp (r =0.69, P <0.0001 with respect to HOMA-IS and r =0.72, P <0.0001 with respect to QUICKI).

Insulin Sensitivity in Patients with Liver Cirrhosis

When analysis was performed separately in patients with liver cirrhosis, fasting plasma glucose was significantly associated with both GIR (r =−0.63, P <0.005) and SIclamp (r =−0.61, P <0.007). On the contrary, fasting plasma insulin was not significantly related to GIR (r =−0.06, P =0.76) or SIclamp (r =−0.25, P =0.32). GIR was significantly associated with HOMA-IS (r =0.48, P =0.05) and QUICKI (r=0.43, P =0.07). These relationships improved when SIclamp was used as estimate of insulin sensitivity (Fig. 1:r =0.63, P <0.01 and r =0.60, P <0.01 with respect to HOMA-IS and QUICKI, respectively). When analyzed separately in patients with both liver cirrhosis and diabetes, the association of SIclamp with HOMA-IS (r =0.51, P <0.05) and QUICKI (r =0.47, P =0.05) remained significant. When tested in the nondiabetic patients with liver cirrhosis, the association of SIclamp with HOMA-IS (r =0.86, P =0.01) and QUICKI (r =0.87, P <0.01) was apparently stronger.

F1-16
Figure 1:
The relationships between the SIclamp (x axis) with HOMA-IS (left) and QUICKI (right) are graphically summarized in patients with liver cirrhosis (upper panels), liver transplant patients (middle panels), and normal individuals (lower panels).

Insulin Sensitivity in the LTx Patients

When analysis was performed separately in LTx patients, fasting plasma glucose was inversely associated with both GIR (r =−0.52, P <0.001) and SIclamp (r =−0.63, P <0.0001). Fasting plasma free-insulin was inversely associated with GIR (r =−0.34, P =0.04) but not with SIclamp (r =−0.25, P =0.15). GIR was significantly associated with HOMA-IS (r =0.49, P =0.003) and QUICKI (r =0.53, P <0.001). When SIclamp was used as a clamp-derived estimate of insulin sensitivity, the same trend was observed (Fig. 1:r =0.41, P =0.02 and r =0.46, P <0.01 with respect to HOMA-IS and QUICKI, respectively). When analyzed separately in liver transplant patients with diabetes, the association of SIclamp with HOMA-IS (r =0.42, P <0.1) and QUICKI (r =0.44, P =0.08) showed a trend to be significant. When tested in the nondiabetic transplant patients, the association of SIclamp with HOMA-IS (r =0.52, P =0.03) and QUICKI (r =0.57, P <0.02) was significant.

Insulin Sensitivity in Healthy Individuals

When analysis was performed separately in normal individuals, fasting plasma glucose was not associated with GIR (r =−0.26, P =0.17) or SIclamp (r =−0.01, P =0.99); on the contrary, fasting plasma free-insulin was significantly associated with both GIR and SIclamp (r =−0.66, P <0.0001 and r =−0.71, P <0.0001, respectively). GIR was significantly associated with HOMA-IS (r =0.69, P <0.0001) and QUICKI (r =0.71, P <0.0001). When SIclamp was used as clamp-derived estimate of insulin sensitivity, the same trend was observed (Fig. 1:r =0.77, P <0.0001 and r =0.77, P <0.0001 with respect to HOMA-IS and QUICKI, respectively).

Insulin Sensitivity in Patients with Liver Cirrhosis Before and 2 Year After LTx (Longitudinal Study)

The 10 patients with liver cirrhosis were studied before LTx and 25±4 months after the surgical procedure. After LTx, fasting plasma free-insulin (P <0.001) concentration was significantly reduced. Both clamp-derived and fasting blood sample-derived parameters of insulin sensitivity (GIR, SIclamp, HOMA-IS, and QUICKI) significantly improved after LTx with respect to the pretransplantation condition (Table 2). In Figure 2 the temporal changes of SIclamp (left), HOMA-IS (middle), and QUICKI (right) are depicted.

F2-16
Figure 2:
The improvement of insulin sensitivity from the pretransplantation condition (Pre) to the posttransplantation condition (Post) is graphically summarized for the SIclamp (dL/[min·kg]/(μU/mK) × 104), HOMA-IS, and QUICKI. Single individuals (filled circles); average ± SD (open circles).

DISCUSSION

Insulin resistance is a constant feature of patients with chronic liver diseases. It is recognized that critical prognostic factors markedly affecting the natural history of liver cirrhosis (hepatocellular failure and impairment of energy balance) are influenced by the insulin-resistant state. Because a classical clamp-derived assessment of insulin sensitivity is not feasible for routine clinical use in patients with liver diseases, we assessed whether simpler surrogate measures of insulin sensitivity based on a single blood sample could be adopted. The aim of this work was twofold: (1) to test whether HOMA-IS and QUICKI may be useful tools to assess insulin sensitivity in patients with liver cirrhosis and in liver transplant patients; and (2) to assess whether these indices are able to prospectively monitor changes of insulin sensitivity induced by LTx.

This work showed that in patients with Child-Pugh A-B liver cirrhosis and in liver transplant patients, both HOMA-IS and QUICKI were reliable surrogates of insulin sensitivity (Fig. 1). Interestingly, in both groups, fasting plasma glucose concentration was more informative than fasting plasma insulin concentration: this was a result of the fact that the prevalence of diabetes in these groups was 61% and 44%, respectively, allowing fasting plasma glucose concentrations to span a wide range. When diabetic individuals were excluded (data not shown), fasting plasma insulin concentration became by far the most informative variable. In keeping with this finding, in healthy nondiabetic individuals, fasting plasma insulin concentration was associated with GIR and SIclamp; meanwhile, fasting plasma glucose concentration was not as useful.

Recently, it has been shown that QUICKI successfully predicted changes of insulin sensitivity after administration of insulin sensitizer drugs (20) or combined administration of diet and exercise training programs in patients with type 2 diabetes (21) and changes of insulin sensitivity in patients with essential hypertension after drug treatment (22). Thus, we wanted to test whether HOMA-IS and QUICKI were useful to longitudinally monitor changes of insulin sensitivity in patients with liver cirrhosis. LTx was previously shown to considerably improve glucose homeostasis (3,16,23), and for this reason it was expected to determine a significant change in the degree of insulin resistance. Indeed, SIclamp improved in all 10 patients participating to the prospective study (Fig. 2, left), and HOMA-IS and QUICKI were all able to detect a similar improvement in all subjects but one (Fig. 2, middle and left, respectively). As can be seen in Table 2, after LTx the improvement of insulin sensitivity was able to completely normalize fasting plasma glucose and free-insulin; therefore, in addition to SIclamp, HOMA-IS and QUICKI were also completely normalized. Future study will tell whether HOMA-IS and QUICKI are able to reveal less dramatic improvements of insulin sensitivity in patients with liver cirrhosis, such those expected after body weight reduction or a light exercise training program. It will be interesting also to test whether these indices may be reliable surrogates of insulin resistance in more prevalent chronic liver diseases such as nonalcoholic fatty liver disease or nonalcoholic steatohepatitis.

Recently our group showed that a revised QUICKI, incorporating the logarithm and the reciprocal of plasma free-fatty acid (FFA) concentration to the insulin-glucose product, performed better than QUICKI in normal individuals, in subjects with moderate alterations of insulin sensitivity (24), and in particular in kidney-pancreas transplant patients (25). In the present work, the revised QUICKI was confirmed to better reflect the clamp-based index of insulin sensitivity in the group of healthy control individuals (predominantly men who were older and fatter than the normal subjects that were examined in ref. 24). The revised QUICKI also improved the association with insulin sensitivity in the group of LTx patients (r =0.62, P <0.0001). In contrast, its performance was not as good as HOMA-IS and QUICKI in the group of patients with liver cirrhosis. A likely explanation for this result is that there was no association between the fasting plasma FFA levels and SIclamp, because in patients with liver cirrhosis plasma FFA concentrations were high and confined within a narrow range.

The use of HOMA-IS and QUICKI as surrogates of insulin sensitivity assessment in studies involving a large number of patients with chronic liver diseases is more convenient than the insulin clamp because of the following reasons: (1) It does not require trained personnel to operate the clamp. (2) It uses a blood sample of 4 to 5 ml instead of the 100 to 130 ml necessary on average for a clamp study. (3) It requires only the time for a venipuncture instead of the 3 to 5 hr necessary for the execution of a clamp study. (4) It minimizes the patient’s discomfort. (5) It drastically reduces the costs associated with assay measurement to more than 1/15 of the cost of the clamp study.

CONCLUSION

This study represents the first attempt to assess analytically whether surrogate indices of insulin sensitivity obtained from measurements of plasma glucose and insulin in a fasting single blood sample may be a reliable measure of clamp-based quantification of insulin resistance in patients with liver cirrhosis before and after LTx. The results obtained in these patients showed that HOMA-IS and QUICKI may be good tools in both cross-sectional and prospective studies.

Acknowledgments.

The authors thank Antonella Scollo, R.N., of the Metabolic Unit of the Istituto Scientifico H San Raffaele for nursing assistance and the Hepato-Pancreatic Surgery and Liver Transplantation Unit of the National Cancer Institute for excellent assistance.

REFERENCES

1. Megyesi C, Samols E, Marks V. Glucose tolerance and diabetes in chronic liver disease. Lancet 1967; 2: 1051–1056.
2. Kruszynska YT, Home PD, McIntyre N. Relationship between insulin sensitivity, insulin secretion and glucose tolerance in cirrhosis. Hepatology 1991; 14: 103–111.
3. Perseghin G, Mazzaferro V, Piceni Sereni L, et al. Contribution of reduced insulin sensitivity and secretion to the pathogenesis of hepatogenous diabetes: effect of liver transplantation. Hepatology 2000; 31: 694–703.
4. Blendis L, Brill S, Oren R, et al. Hepatogenous diabetes: reduced insulin sensitivity and increased awareness. Gastroenterology 2000; 119: 1800–1802.
5. Perseghin G, Mazzaferro V, Benedini S, et al. Resting energy expenditure in diabetic and non-diabetic patients with liver cirrhosis: relationship with insulin sensitivity, and effect of liver transplantation and of immunosuppressive therapy. Am J Clin Nutr 2002; 76: 541–548.
6. Clark JM, Brancati FL, Diehl AE. Nonalcoholic fatty liver disease: the most common cause of abnormal liver enzymes in the US population [abstract]. Gastroenterology 2000; 14: 321–326.
7. Marchesini G, Brizi M, Bianchi G, et al. Nonalcoholic fatty liver disease: a feature of the metabolic syndrome. Diabetes 2001; 50: 1844–1850.
8. Yu AS, Keefe EB. NAFLD and NASH: important diseases before and after liver transplantation. Hepatology Elsewhere 2001; 34: 842–843.
9. De Fronzo RA, Tobin JD, Andres R. Glucose clamp technique: a method for quantifying insulin secretion and resistance. Am J Physiol 1979; 6: E214–E223.
10. American Diabetes Association: Consensus Development Conference on Insulin Resistance. Diabetes Care 1998; 21: 310–314.
11. Bergman RN. Lilly Lecture 1989. Toward physiological understanding of glucose tolerance: minimal model approach. Diabetes 1989; 38: 1512–1527.
12. Matthews DR, Hosker JP, Rudenski AS, et al. Homeostasis model assessment: insulin resistance and β-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 1985; 28: 412–419.
13. Bonora E, Targher G, Alberiche M, et al. Homeostasis model assessment closely mirrors the glucose clamp technique in the assessment of insulin sensitivity. Diabetes Care 2000; 23: 57–63.
14. Katz A, Nambi SS, Mather K, et al. Quantitative insulin sensitivity check index: a simple, accurate method for assessing insulin sensitivity in humans. J Clin Endocrinol Metab 2000; 85: 2402–2410.
15. Radziuk J. Insulin sensitivity and its measurement: structural commonalities among the methods. J Clin Endocrinol Metab 2000; 85: 4426–4433.
16. Perseghin G, Scifo P, De Cobelli F, et al. Intramyocellular triglyceride content is a determinant of in vivo insulin resistance in humans: a 1H-13C NMR spectroscopy assessment in offspring of type 2 diabetic parents. Diabetes 1999; 48: 1600–1606.
17. Perseghin G, Regalia E, Battezzati A, et al. Regulation of glucose homeostasis in humans with denervated livers. J Clin Invest 1997; 100: 931–941.
18. Bergman RN, Finegood DT, Ader M. Assessment of insulin sensitivity in vivo. Endocrine Rev 1985; 6: 45–86.
19. Caumo A, Simeoni M, Cobelli C. Models of glucose and insulin control on glucose metabolism. In: Carson E, Cobelli C, eds. Modelling methodology for physiology and medicine, chap. 12. San Diego, Academic Press, 2001, pp 337–372.
20. Mather KJ, Hunt EH, Steinberg HO, et al. Repeatability characteristics of simple indices of insulin resistance: implications for research applications. J Clin Endocrinol Metab 2001; 86: 5457–5464.
21. Katsuki A, Sumida Y, Gabazza EC, et al. QUICKI is useful for following improvements in insulin sensitivity after therapy in patients with type 2 diabetes mellitus. J Clin Endocrinol Metab 2002; 87: 2906–2908.
22. Chen H, Sullivan G, Yue LQ, et al. QUICKI is a useful index of insulin sensitivity in subjects with hypertension. Am J Physiol 2003; 284: E804–E812.
23. Luzi L, Perseghin G, Regalia E, et al. Metabolic effects of liver transplantation in cirrhotic patients. J Clin Invest 1997; 99: 692–700.
24. Perseghin G, Caumo A, Caloni M, et al. Incorporation of the fasting plasma FFA concentration into QUICKI improves its association with insulin sensitivity in nonobese individuals. J Clin Endocrinol Metab 2001; 86: 4776–4781.
25. Perseghin G, Caumo A, Piceni Sereni L, et al. Fasting blood sample-based prediction of insulin sensitivity in kidney-pancreas transplanted patients. Diabetes Care 2002; 25: 2207–2211.
© 2003 Lippincott Williams & Wilkins, Inc.