Protein, glucose and lipid metabolism in the cancer cachexia: A preliminary report

To the Editor

Cancer cachexia is a complex syndrome characterized by weight loss with depletion of both skeletal muscle and adipose tissue mass, associated with extreme weakness, dry and wrinkled skin. Cancer cachexia is associated with protein degradation of human skeletal muscle with a slow or fast progression rate.

The mechanism of wasting occurring in malignant diseases is multifactorial. The causes of the cancer cachexia syndrome may include decreased food intake [1], increased faecal and urinary nutrient losses, and malfunction of metabolic pathways in both the host and the tumour. Also the site and the mass of the tumour may be involved in causing anorexia due to excessive metabolites consumption. In addition metabolic alterations induced by the tumour itself, through production of specific factors (several cytokines, especially TNF-α, INF-γ, and IL-6), may be interfering with normal metabolism [2].

Glucose intolerance is one of the earliest metabolic alterations recognized in patients with cancer [3]. The whole body glucose turnover is increased, while glucose utilization is impaired due to insulin resistance. Increased gluconeogenesis from lactate and alanine is also a typical alteration of glucose metabolism in cancer cachexia [4]. Insulin resistance does not appear to be a consequence of malnutrition, but seems to be related to the tumour itself [5].

Depletion of muscle protein masses is typical in cancer cachexia with increased tumour protein accumulation [2]. The majority of the authors have found increased whole body protein turnover [6], while the results on protein synthesis (enhanced, decreased) and on the relative resistance to insulin [7] are not consistent.

As the progressive loss of muscle mass is the most prominent phenotypic feature of cancer cachexia, we studied amino acids metabolism in order to help to develop more effective therapeutic strategies for preventing cancer-related muscle wasting.

With this purpose we assessed insulin action on protein, lipid and glucose metabolism in patients affected by cancer with a recent relevant weight loss, utilizing the classical technique of the euglycemic hyperinsulinemic clamp which is the golden standard procedure for the determination of insulin action.

All patients were recruited in the Department of Medicine of S. Raffaele Hospital, Milano, Italy. Five patients (3 males and 2 woman, age: 61±17 years; BMI: 20.5±3.9 kg/m²) (CC) affected by cancer (two patients with lung cancer, one patient with gastric cancer and two women with ovary cancer) with a mean 12% weight loss were studied in the last three months. Ten lean healthy volunteers (CON) with stable normal weight were also studied as control group (7 males and 3 female, age: 55±8 years; BMI: 22.1±1.2 kg/m²).

Patients and volunteers were fully informed of the possible risks of the study and gave their consent. The Ethical Committee of the Istituto Scientifico H. San Raffaele approved the experimental protocol. All studies were performed, while the patients were hospitalized, in the postabsorptive state after an overnight fast.

The euglycemic hyperinsulinemic clamp was performed as previously described [8] associated with a prime-continuos infusion of (3-³H) glucose and (1-¹⁴C) leucine to assess rates of glucose and protein metabolism. After a 150 minutes equilibration period, insulin was infused at the rate of 1 mU/kg/min; plasma glucose was maintained at the basal level by a 20% glucose infusion. Insulin infusion was continued for 150 minutes. Arterialized blood samples were drawn every 15 minutes to assess glucose and leucine specific activities and every 30 minutes to evaluate hormone concentrations (free-insulin, C-peptide, glucagon, cortisol, IGF-1 and growth hormone) and intermediate metabolites (amino acids, lactate, pyruvate, NEFA and glycerol). Indirect calorimetry was performed for 30 minutes during the basal condition and at the end of the hyperinsulinemic period to calculate metabolite rates of oxidation [9].

Plasma glucose was measured bedside with a Beckman Glucose Analyzer. Free-insulin, C-peptide, glucagon, cortisol, IGF-1 and growth hormone were measured by commercial RIA kits. Lactate, pyruvate, NEFA and glycerol were dosed by an automated immunofluorimetric assay. Plasma leucine and alanine concentrations were determined by HPLC as DABS-derivatives. Tritiated glucose and ¹⁴C-leucine specific activities were measured in plasma as previously described [9]. ¹⁴CO₂-specific activity in breath was measured by ß scintillation as described [9]. Kinetics were calculated for the last 60 minutes of both the equilibration and the hyperinsulinemic periods when steady-state conditions were reached. The data of protein turnover were derived from values of plasma leucine specific activity (primary pool) using a stochastic model as previously reported [10]. Data are given as mean ±SE. Comparisons between basal and insulin stimulated state within a group were performed with t-test for paired data. Comparisons between groups were done using independent-samples test.

The fasting plasma glucose was within the normal range in all patients and in the control group (4.9±0.4 vs 4.5±0.1mmol/l, p = ns). In the basal condition no differences between the two groups were detected for plasma C-peptide, glucagon and cortisol concentrations and also NEFA, glycerol, lactate, pyruvate and ß-OH-butyrate were not different. Plasma leucine was similar in CC and CON in the post-absorptive state (123±22 vs 120±20 µmol/l, p = ns).

Plasma free-insulin in the basal condition was significantly higher in CC in comparison to controls (11.1±2.7 vs 5.8±1 µU/ml, p < 0.001). Also GH was increased (basal condition: 1.9±0.6 vs 1.4±0.5, p = 0.08; clamp: 5.0±1.5 vs 2.8±1.9 ng/ml, p < 0.05 in CC and CON respectively), in contrast IGF-1 was lower in the basal condition and after insulin stimulation (152±45 vs 185±39, p < 0.05 and 160±51 vs 196±35 ng/ml, p < 0.05).

Total glucose disposal in the study period was significantly reduced in CC vs CON (3.28±0.7 vs 7.01±0.33 mg/kg/min, p < 0.001). In the postabsorptive state, ELF (endogenous leucine flux, an index of proteolysis) (53.3±22.4 vs 42.5±2.5 µmol/m²/min, p = 0.45), leucine oxidation (LO: 5.7±0.5 vs 6.7±0.7 µmol/m²/min, p = 0.61) and nonoxidative leucine disposal (NOLD an index of protein synthesis) (47.6±9.9 vs 35.8±1.9 µmol/m²/min, p = 0.48) were not different (Table I). Acute hyperinsulinemia induced a much less pronounced decrease of plasma leucine (38%), ELF (21%), LO (14%) in comparison to the controls (52% plasma leucine, 39% ELF, 24% LO, p < 0.05).

Table I.  Parameters of leucine kinetics and glucose metabolism in basal conditions and during clamp in patients with cachexia and control group.

Leucine kinetics
	basal	hyperinsulinemia
Patients
Plasma leucine µmol/l	123.1±22.4	74.5±14.7
ELF µmol/m^2/min	53.3±10.3	41.7±10.4
LO µmol/m^2/min	5.7±0.5	5.0±1.6
NOLD µmol/m^2/min	47.6±9.9	36.7±9.2
Controls
Plasma leucine µmol/l	120.5±2.1	57.6±5.9
ELF µmol/m^2/min	42.5±2.5	25.9±1.4
LO µmol/m^2/min	6.7±0.7	5.1±0.4
NOLD µmol/m^2/min	35.8±1.9	28.8±1.7
Glucose metabolism
Patients
Plasma glucose (mmol/l)	4.93±0.39	4.93±0.40
f-Insulin (µU/ml)	11.1±2.7*	64.3±8.2
Tissue Glucose uptake (mg/kg/min)		3.28±0.67*
Controls
Plasma glucose (mmol/l)	4.47±0.14	4.59±0.45
f-Insulin (µU/ml)	5.8±0.96	61.6±15.3
Tissue Glucose uptake (mg/kg/min)		7.01±0.33

*p < 0.001 vs controls.

(ELF: endogenous leucine flux, LO: leucine oxidation and NOLD: nonoxidative leucine disposal).

Indirect calorimetry data showed increased lipid oxidation in CC in comparison with CON, both in the postabsorptive state (217.19±59.23 vs 94.2±16.8 µmol/m²/min) (p < 0.001) and during hyperinsulinemia (177.78±62.85 vs 29.4±12.9 µmol/m²/min) (p < 0.001). Also lactate and pyruvate after insulin stimulation were higher (p < 0.05 vs CON).

In each cancer patient, the signs of insulin resistance for glucose metabolism were well evident. This is not surprising considering that this event has been described since many years [2], [3] and it is mainly related to the production of specific cytokines by the tumor itself. Actually, insulin resistance has been documented even in patients affected by cancer without evident weight loss [11].

Indirect calorimetry data documented that also lipid metabolism was impaired in our cachectic patients; the insulin-induced suppression of lipid oxidation was impaired (34% vs 58% p < 0.02).

Alterations of insulin mediated protein metabolism were less profound; plasma leucine concentration and ELF were not different in the basal condition. In contrast during hyperinsulinemia the inhibition of proteolysis was markedly reduced and we can not exclude that the defects may be more pronounced if the reciprocal pool model was used instead of the primary pool. In summary insulin action is not fully able to control catabolic processes in the muscle in these patients. A recent report suggests that IGF-1 could be involved into the pathogenesis of cancer cachexia. This hypothesis is sustained by the well known anabolic action of this hormone on skeletal muscle, which is supposed to counteract the muscle wasting as the most important phenotypic feature of cancer cachexia [12]. In agreement with a potential role of IGF-1 in patients with cancer cachexia with respect to controls, this result was found in association with increased plasma GH concentration in either the basal and insulin stimulated conditions. Therefore these results could be interpreted as growth hormone resistance with respect to hepatic IGF-1 synthesis.

These preliminary data could have a strong clinical impact, in fact based on them: 1) patients with cancer cachexia need close monitoring of parameters of the endocrine status and of energetic and nutritional homeostasis 2) these patients require a more aggressive nutritional management to prevent malnutrition (the use of amino acids integrator contain carnitine may be recommended) [13] and then improve the qualify of life in this patients seriously ill.

In conclusion, our preliminary data, demonstrated that insulin resistance plays a role in the complex clinical and metabolic picture of cancer cachexia not only on glucose metabolism, but also on protein and lipid metabolism. Further studies are warranted to better understand the role of insulin resistance into the pathogenesis of cancer cachexia and to define potential novel strategy for its treatment.