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Exp Gerontol. Author manuscript; available in PMC 2010 June 29.
Published in final edited form as:
PMCID: PMC2894083

HISS-dependent insulin resistance (HDIR) in aged rats is associated with adiposity, progresses to syndrome X, and is attenuated by a unique antioxidant cocktail


The hypotheses were: HISS-dependent insulin resistance (HDIR) accounts for insulin resistance that occurs with aging; HDIR is the initiating metabolic defect that leads progressively to type 2 diabetes and the metabolic syndrome; a synergistic antioxidant cocktail in chow confers protection against HDIR, subsequent symptoms of diabetes, and the metabolic syndrome. Male Sprague Dawley rats were tested at 9, 26, and 52 weeks to determine their dynamic response to insulin, the HISS (hepatic insulin sensitizing substance)-dependent component of insulin action, and the HISS-independent (direct) insulin action using a dynamic insulin sensitivity test. In young rats, the HISS component accounted for 52.3 ± 2.1% of the response to a bolus of insulin (50 mU/kg) which decreased to 29.8 ± 3.4% at 6 months and 17.0 ± 2.7% at 12 months. HISS action correlated negatively with whole body adiposity and all regional fat depots (r2 = 0.67–0.87). The antioxidants (vitamin C, vitamin E, and S-adenosylmethionine) conferred protection of HISS action, fat mass at all sites, blood pressure, postprandial insulin and glucose. Data are consistent with the hypotheses. Early detection and therapy directed towards treatment of HDIR offers a novel therapeutic target.

Keywords: Antioxidant vitamins, Age, Regional adiposity, Body weight, HISS

1. Introduction

The presence of a mixed meal in the upper gastrointestinal tract results in a meal-induced insulin sensitization. Feeding results in a doubling of the dynamic glucose disposal response to insulin (Lautt et al., 2001). According to the HISS (hepatic insulin sensitizing substance) hypothesis (reviewed Lautt, 2004), in the early postprandial state, feeding signals delivered to the liver are required in order that pulses of insulin are able to cause the generation and release of a hormone from the liver that acts selectively on skeletal muscle (Xie and Lautt, 1996a,b; Moore et al., 2002) to stimulate glucose uptake. This, as yet unidentified, hormone is referred to as hepatic insulin sensitizing substance (HISS). Both the HISS-dependent insulin action and the HISS-independent component (the direct action of insulin) can be quantified by the dynamic responses to a pulse of insulin (Lautt, 2003).

HISS action accounts for approximately 55% of the total glucose disposal response to pulses of insulin over a wide range of insulin doses (5–100 mU/kg) (Lautt et al., 2001). Two “feeding signals” are required for insulin to cause HISS release. Hepatic glutathione levels are increased by 30–50% soon after feeding and, in the absence of this increase, HISS release is blocked (Guarino et al., 2004). The second feeding signal is delivered by hepatic parasympathetic nerves (Xie and Lautt, 1996a,b), mediated by hepatic nitric oxide (Sadri et al., 1997; Sadri and Lautt, 1999) associated with hepatic cyclic GMP (Correia et al., 2002; Guarino et al., 2004). Meal-induced insulin sensitization can be blocked by any intervention that inhibits these signals and blocks HISS release/actions (Lautt et al., 2001). HISS action can be quantified by comparing the response to insulin in the fed and fasted state, or from insulin action in the fed state before and after blockade of HISS release following, for example, inhibition of hepatic nitric oxide synthase or hepatic cholinergic muscarinic receptors, or hepatic denervation (Lautt et al., 2001). Following hepatic denervation, HISS release can be restored by mimicking the parasympathetic feeding signal through provision of a background dose of nitric oxide (Sadri and Lautt, 1999) or a muscarinic agonist to the liver (Xie and Lautt, 1996a). Meal-induced insulin sensitization can be prevented from occurring by denervating the liver; if sensitization is allowed to develop, it can be completely reversed by atropine administration (Sadri et al., 2006).

HISS release is reduced progressively with the duration of fasting until, by 24 h, HISS release in response to insulin becomes insignificant and results in a state of physiological HISS-dependent insulin resistance (HDIR). HDIR is biologically appropriate in the fasted state to prevent insulin-induced hypoglycemia. However, if it occurs in the fed state, it has been suggested to account for postprandial hyperglycemia and compensatory hyperinsulinemia. The increased insulin acts primarily on adipose tissue and liver and results in nutrient storage shifting from glycogen in skeletal muscle to lipid, thus resulting in a lipogenic state and increased production of reactive oxygen species (Lautt, 2004).

Age is associated with an increased risk of insulin resistance and type 2 diabetes (Singh and Marshall, 1995; Dela and Kjaer, 2006). Aging is also associated with a decrease in lean body mass and a relative increase in fat mass and has been suggested to result in a slow progressive redistribution of fat from subcutaneous tissue to central abdominal tissue (Borkan et al., 1993; Barbieri et al., 2001). Aging in Wistar rats results in insulin resistance that can be accounted for by HDIR (Ribeiro et al., in press). The HISS component was reduced from 53% of the total response to 18% at 1 year. Aging is also associated with a generalized reduction in parasympathetic nerve function demonstrated for the cardiovascular system (O’Brien et al., 1986; Ingall et al., 1990; Corbett et al., 2007), eyes (Fitzgerald et al., 2005; Tewari et al., 2006; Jablonski et al., 2007), GI tract (Phillips and Powley, 2001, 2007), and urinary bladder (Schneider et al., 2005). Parasympathetic dysfunction can be identified prior to development of diabetes (Lautt, 1980), and is later associated with the “metabolic syndrome” (Britton et al., 2007) and obesity (Skrapari et al., 2007; von Kanel et al., 2007) and improves with reduced adiposity (Mager et al., 2006).

While adiposity and insulin resistance are well recognized to be related, which causes which remains controversial (reviewed Robinson and Graham, 2004) as does the relationship between insulin resistance and regional adiposity. Many studies claiming a relationship of abdominal obesity and insulin resistance measured only abdominal obesity or its surrogate indicator, waist circumference. These studies imply but do not contribute to the issue of regional adipose selective roles. While a special role has been proposed for adipose tissues that release lipid directly to the liver via the portal vein (Bjorntorp, 1992; Goran et al., 2001; Lebovitz, 2003; Lindmark et al., 2005), others have failed to show any special role for this portal theory (Frayn, 2000) or for selective regional adiposity (Goodpaster et al., 1997; Taniguchi et al., 2002; Tulloch-Reid et al., 2004). In the present study we examine the relationship of HDIR to regional adiposity.

The free radical theory of aging, first proposed by Harman (1956), suggests that endogenous free radicals continuously and progressively cause permanent mitochondrial damage (Sastre et al., 1996; Cadenas and Davies, 2000). The capacity to scavenge reactive oxygen species decreases with age (Esteve et al., 1999; Rebrin et al., 2004). While insulin resistance results in excess free radicals, reactive oxygen species are also proposed as a trigger for insulin resistance in numerous settings (Houstis et al., 2006). We have recently shown that a hepatotoxin, thioacetamide, known to produce liver damage by generation of reactive oxygen species, results in HDIR that can be prevented by a synergistic combination of antioxidants simultaneously targeting the aqueous phase (vitamin C), the lipid phase (vitamin E), and the mitochondria (S-adenosylmethionine). This combination, referred to for convenience as Samec, confers dramatic synergy as shown by efficacy of the combination but lack of protection by the individual components (Ming et al., 2006). Its effects are evaluated in this study.

An objective of this project was to determine if insulin resistance occurs with aging up to 1 year in Sprague Dawley rats and to assess to what extent the insulin resistance is accounted for by HDIR. We test the hypotheses that HDIR leads progressively to generalized adiposity and symptoms of diabetes and the metabolic syndrome. The role of regional adiposity was determined by weighing fat mass in the perinephric and epididymal fat pads, which drain to the vena cava, and the perienteric fat mass, which drains to the portal vein; whole body adiposity was assessed using bioelectrical impedance (Hall et al., 1989). We tested the effect of the antioxidant cocktail, administered through the rat chow, to attenuate the development of HDIR and adiposity with age.

2. Materials and methods

The experimental procedures were approved by an ethics committee on animal care at the University of Manitoba and performed in accordance with the Guidelines of the Canadian Council on Animal Care.

2.1. Animals and groups

Male Sprague Dawley rats (Charles River, St. Constant, Quebec, Canada) 7 weeks old (body weight 200–225 g) were maintained under controlled conditions (22 ± 1 °C, 12 h light/12 h dark cycle). They were fed with a standard rat chow diet (Prolab RMH300, PMI Feeds, St. Louis, MO) with free access to water for 2 weeks to adapt to the housing environment. Then the animals were divided into two groups, one fed with normal chow; the other with normal chow supplemented with antioxidants (SAMe (0.5 g/kg diet), vitamin C (12.5 g/kg diet), and vitamin E (1.5 g/kg diet)). The supplemented chow was formulated with the drugs, vacuum purged with nitrogen and sealed in foil bags (Research Diets Inc., New Brunswick, NJ). Given the average daily food consumption of 20 g, the approximate daily intake for vitamin C is 250 mg/kg body weight, for vitamin E is 30 mg/kg body weight and for SAMe is 19 mg/kg body weight. Rats, for ethical reasons, were housed in pairs.

Rats included in these two groups were tested at the ages of 6 and 12 months (n = 13/group). Young adult rats (n = 8) at the age of 9 weeks, fed with standard rat chow, served as the young control group for 6 and 12 month old rats.

2.2. Surgical preparation

All rats underwent an 8 h fast and a refeeding period of 2 h immediately before the start of surgical preparation. Because the HISS component of insulin action is only seen in the fed state, the fasting-refeeding protocol assures a high level of HISS release in response to insulin. The rats were anaesthetized with an intraperitoneal injection of sodium pentobarbital (65 mg kg−1; Somnotol, MTC Pharmaceuticals, Ont.). Anesthesia was maintained by a continuous infusion of pentobarbital sodium (0.5 mg ml−1 saline given at 50 μl min−1) through a cannula in the jugular vein, supplemented with a 0.65 mg (0.01 ml) bolus injection when required. The rats were placed on a homeothermic temperature-controlled surgical table (Harvard Apparatus, Kent, England) and rectal temperature was monitored and held at 37–37.5 °C. Spontaneous respiration was allowed through a tracheal catheter.

An arterial-venous shunt was established, as previously described (Lautt et al., 1998), for monitoring mean arterial blood pressure (MAP), blood glucose level, and for intravenous drug delivery. Briefly, two catheters (polyethylene tubing PE60), one inserted into the right femoral artery and the other into the right femoral vein, were connected with silicon tubing. A side branch of the circuit was connected to a pressure transducer for recording shunt pressure. When the silicon tubing toward the venous side of the circuit was closed by clamping, the transducer measured systemic arterial blood pressure. Blood samples were taken from the arterial side of the loop for the glucose measurement. Flowing blood within the loop assures the real-time measurement of the blood glucose concentration, which is essential for the dynamic euglycemic clamp test as mentioned below. An infusion line was inserted into the venous side of the loop for drug delivery. The infusion line connected to the jugular vein was established for glucose infusion. Animals were heparinized (100 IU kg−1) to prevent clotting in the vascular loop.

2.3. Rapid insulin sensitivity test (RIST)

The RIST was performed as previously described (Lautt et al., 1998). Briefly, following completion of surgery, animals were allowed at least a 30-min stabilization period. The baseline glucose levels were then determined by samples taken at 5-min intervals and continued until three successive stable determinations were made. The mean of these three data points was regarded as the baseline for the RIST. To perform the RIST, human insulin (50 mU kg−1 in 0.5 ml saline) was infused into the femoral vein at the rate of 0.1 ml min−1 for 5-min. After 1-min of insulin infusion, the first test glucose sample was determined and a variable glucose infusion (10%) was initiated. Blood samples were taken every 2-min and the glucose infusion rate was adjusted to maintain euglycemia. The RIST index was the amount of glucose (mg kg−1) infused, to maintain euglycemia, over the test period which was terminated when no further glucose infusion was required (approximately 35 min). At the end of a RIST, the animal was at its pretest glycemic level.

Two RISTs were performed for each rat. Following the establishment of the glucose baseline, the first RIST index was determined and this was regarded as the control (fed) RIST. The second RIST was performed after blockade of HISS release with intravenous infusion of atropine (1 mg kg−1 in 0.5 ml saline, 0.1 ml min−1) and reestablishment of a stable glycemic baseline. The first RIST index includes the effects of both HISS-dependent and HISS–independent components, and the second RIST index represents only the HISS-independent component (reviewed by Lautt, 2003). The percentage contribution of HISS-dependent component to total insulin sensitivity was calculated as: % HISS = (RIST index in control − RIST index after atropine)/RIST index in control × 100%.

2.4. Tissue and blood samples

Whole body fat mass (FM) was estimated by using electrical impedance measurement (Hall et al., 1989). FM (%) was calculated as the ratio of FM to body weight × 100%. Visible fat tissue of the perinephric fat pad, epididymal fat pad and perienteric fat pad were collected and weighed. The sum of these three fat masses was termed fat pad mass and its ratio to body weight termed % fat pad mass.

2.5. Data collection, instruments, and chemicals

A data acquisition system combined with computer application software (National Instruments Lab View, Austin, Texas), was used to record and analyze the mean arterial blood pressure and to provide real-time monitoring of progression of the RIST and quantification of adherence to the euglycemic target baseline (% precision and accuracy). A RIST was discarded if the euglycemic baseline was missed by more than 5% at any time point. Blood glucose concentration was measured by a glucose analyzer (Yellow Springs Instrument, Yellow Springs, Ohio). The infusion pumps were from Kent Scientific Corporation, Torrington, CT (Model RS 232). Electrical impedance measurement for body fat mass estimation was measured using a Bioelectrical Body Composition Analyzer (RJL systems, Clinton Twp., MI, USA).

Human insulin was purchased from Novo Nordisk (Mississauga, Ont.). Atropine, vitamin C (L-Ascorbic acid), vitamin E ((±)-α-Tocopherol), and SAMe were all purchased from Sigma. Insulin and atropine were dissolved in saline. The Samec formulation was incorporated into regular rat chow (Research Diets Inc., New Brunswick, NJ). Insulin was assayed by ELISA (ALPCO, Windham, NH).

2.6. Statistical analysis

Values are presented as means ± SE. The data were analyzed by paired or unpaired t-test where appropriate. A one-way ANOVA followed by Tukey’s test was employed when the multiple means from different groups were compared. Statistical significance was taken at P < 0.05. Linear regression was used.

3. Results

3.1. Development of obesity with aging

3.1.1. Bioelectrical impedance analysis of whole body fat

Data are summarized in Table 1. Compared to young rats, 6 and 12 month old rats gained body weight by about 86% and 124%, respectively. However, the whole body fat gain was dramatically disproportionately greater than body weight gain, increasing to 381% at the age of 6 months and further to 752% at 12 months, resulting in an increment in percent of body weight as fat (% FM). In the young rats fat accounted for 8.6 ± 0.5% of total body weight and this proportion increased to 22.5 ± 1.4% at 6 months and further to 33.1 ± 1.7% at 12 months.

Table 1
Effects of aging and antioxidant treatment on the development of obesity in rats

3.1.2. Fat pad accumulation

The total fat pad mass, including perinephric, perienteric, and epididymal FM, increased significantly with age, as reported in Table 1 and Fig. 1. The rate of fat accumulation tended to be greater in the perinephric fat pad, increasing by 459% at 6 months and further to 889% at 12 months. Perienteric FM also increased quickly, by 379% and 652% in 6 and 12 month old rats. Epididymal FM increased quickly by 6 months (368% of young rats) and slowed down thereafter, only reaching to 468% at 12 months.

Fig. 1
Aging on regional fat accumulation. Aging was associated with non-selective fat accumulation and antioxidant treatment partially prevented this tendency. 6 or 12, 6 or 12 month old rats; C or V, control or antioxidant diet. FM, fat mass, absolute weight ...

3.1.3. Correlation of whole body fat with weighed fat pads

The relationship between whole body FM, estimated by the impedance method, and total fat pad mass are presented in Fig. 2. The data were pooled from all animals tested. Whole body FM and total weighed FM were directly linearly correlated with data expressed as a % of body weight (r2 = 0.94; P < 0.0001) or absolute weight (r2 = 0.87; P < 0.0001). The weight of each fat pad was also strongly correlated with FM calculated from bioelectrical impedance (r2 = 0.91 perinephric, 0.84 epididymal, 0.91 perienteric) (Fig. 3). These data validate the impedance method.

Fig. 2
Relationship between total body fat mass (FM) as well as % total body FM estimated by the bioelectrical impedance method and total collected visceral FM as well as % visceral FM. Data were from all five rat groups.
Fig. 3
Relationship between body fat mass (FM) estimated by the bioelectrical impedance method and collected individual visceral fat, including perinephric, epididymal, and perienteric FM. Data were from all five rat groups.

3.1.4. Effect of Samec

Intake of Samec did not affect body weight gain with aging (Table 1) but showed the effect of reducing adiposity thus preserving lean body mass. At the age of 6 months, rats receiving Samec from the diet showed a tendency to have less whole body and total weighed FM and % FM as compared to their age-matched partners, although the differences did not reach statistical significance. At the age of 12 months, the whole body % FM decreased from 33.1 ± 1.7% to 28.4 ± 1.6% (P = 0.055, Table 1), and total weighed % FM decreased from 11.0 ± 0.5% to 9.3 ± 0.5% (P < 0.05, Table 1, Fig. 1).

3.2. Aging on MAP, blood glucose, and plasma insulin

The MAP slightly increased with aging and the increase reached significance at the age of 12 months, and this tendency was inhibited by Samec (Table 1). The 2 h postprandial plasma glucose levels increased at 12 months and Samec treatment blunted the increment. Postprandial insulin was elevated with age and Samec provided significant protection in the 12 month groups (Table 1).

3.3. Aging on insulin sensitivity

Young rats had a RIST index of 174 ± 7.3 mg kg−1. Atropine at the dose of 1 mg kg−1 significantly decreased the RIST index to 80.3 ± 2.5 mg kg−1, indicating 52.3 ± 2.1% of insulin sensitivity was attributable to the HISS-dependent component (Fig. 4). Aging progressively decreased insulin sensitivity in rats. As shown in Fig. 4, the RIST index decreased to 100.3 ± 6.1 mg kg−1 in 6 month old rats (P < 0.01 vs young rats) and further decreased to 79.1 ± 4.0 mg kg−1 in 12 month old rats (P < 0.01 vs 6 month old rats). The RIST indexes after atropine administration were 68.4 ± 3.0 mg kg−1 and 64.8 ± 1.9 mg kg−1 in 6 and 12 month rats, respectively, representing the HISS-independent component and indicating that HISS-dependent insulin action only represented 29.8 ± 3.4% and 17.0 ± 2.7% of the response to insulin in these two groups of aging rats. There was also a slight decrease in HISS-independent insulin sensitivity in aged rats, as demonstrated by the post-atropine RIST index in 6 and 12 month old rats being slightly but significantly lower than that observed in young rats (Fig. 4).

Fig. 4
RIST index before and after atropine (top) and % HISS component (bottom) in rats of different ages. RIST index decreased progressively with aging before atropine. RIST index after atropine also slightly decreased in aged rats, but was not different between ...

Atropine administration did not change the basal glucose level (114.3 ± 2.0 mg% before and 110 ± 7.4 mg% post-atropine).

3.3.1. Samec on age-related deterioration in insulin sensitivity

Aging rats receiving Samec from the diet showed improved HISS-dependent insulin sensitivity. As shown in Fig. 5, the RIST indexes were 131.8 ± 9.1 mg kg−1 and 122.2 ± 6.2 mg kg−1, and the HISS component was 43.8 ± 2.1% and 40.7 ± 2.3%, respectively, in 6 and 12 month old rats receiving Samec. The responses to insulin were significantly higher than the age-matched groups that had not received Samec. However, the post-atropine RIST indexes remained similar in rats with or without Samec, indicating that the protection of insulin sensitivity was due to preservation of the HISS-dependent component (Fig. 5).

Fig. 5
Antioxidants on aging-induced reduction in insulin sensitivity. The reduced HISS-dependent insulin sensitivity was greatly prevented by antioxidants. 6 M or 12 M, 6 or 12 month old rats; + Vit, rats treated with antioxidants. *P < 0.01 vs same ...

3.4. Correlation of insulin sensitivity and body fat mass

The relationship between % whole body FM (as measured by bioelectrical impedance) and fat pad mass as a % of body weight and insulin sensitivity was evaluated by pooling the data from all rats receiving normal diet or Samec supplement (Figs. 68). In rats receiving normal food, the RIST index declined with the increase in % whole body FM or fat pad mass, indicating a significantly strong negative correlation. Similar correlations are shown (Fig. 9) for each regional fat pad with no notable regional differences. The slope of the curve indicates that the perinephric fat pad accumulates fat at a greater rate and epididymal at the slowest rate. Provision of Samec reduced the regression coefficients.

Fig. 6
The effect of insulin sensitivity determined from the RIST index on % whole body fat mass and total fat pads as a % of body weight in rats with normal diet or diet containing antioxidants. The slopes of the regression line are similar for the two rat ...
Fig. 8
The effect of HISS-independent insulin action on % whole body fat mass (FM) and total fat pad mass (% of body weight) in rats with normal diet or diet containing antioxidants.
Fig. 9
Effect of antioxidants on correlation between HISS-dependent insulin action and various fat pads expressed as % body weight in rats from 9 to 12 weeks old. The correlation was not changed by receiving antioxidants. N = 39 for both groups.

4. Discussion

This series of experiments was designed to test several interrelated hypotheses. (1) HISS-dependent insulin resistance (HDIR) accounts for insulin resistance that occurs with aging. Our results support this hypothesis and are consistent with the data first reported by Ribeiro et al. (in press). (2) HDIR leads to increased adiposity in general without major regional preferential impact. The data are consistent with this hypothesis, although the perinephric pad showed the largest increase affected by HDIR. These data are consistent with those reporting the effect of a high fat diet on HDIR and obesity (Afonso et al., 2007a). (3) HDIR represents a prediabetic state that progresses to diabetes and the metabolic syndrome. The data are consistent with this hypothesis. At 1 year the rats had developed elevated postprandial insulin and glucose, generalized adiposity, severe HDIR, and elevated blood pressure. However, although the full syndrome was clearly progressing, the rate of development of HDIR and the duration of HDIR impact were insufficient to result in a severe syndrome. Further the mild elevation of postprandial glucose and insulin suggests, but does not demonstrate, progression to frank diabetes. (4) A targeted antioxidant cocktail can attenuate HDIR associated with age. The combination of antioxidants, referred to as Samec, designed to simultaneously protect against reactive oxygen species in the aqueous phase, lipid phase, and mitochondria attenuated HDIR, hypertension, postprandial insulin and glucose, and generalized adiposity.

4.1. The rapid insulin sensitivity test (RIST)

HISS action is defined, for present purposes, as the mechanism of the increase in the dynamic response to insulin that occurs in response to a meal, that is, the mechanism of meal-induced insulin sensitization. Only two current methods are capable of determining a dynamic response to pulses of insulin in both the fed and fasted state without complex modeling. The dynamic glucose disposal response to insulin can be evaluated from the first 15 min of an insulin tolerance test, or from the entire duration of action of a pulse of insulin using the RIST (Reid et al., 2002).

The RIST is a frequently sampled euglycemic clamp carried out in response to an intravenous bolus of insulin. It measures the whole body dynamic response to insulin, in contrast to the fasted, steady-state surrogate indexes of insulin action, which includes the 3 h hyperinsulinemic euglycemic clamp, the HOMA, and QUICKI. The RIST methodology is fully described (Lautt et al., 1998) and methods to quantify HISS-dependent and HISS-independent insulin action have been reviewed (Lautt, 2003). Typically, the glucose disposal effect of a pulse of insulin reaches a peak at approximately 15 min and insulin action is terminated by 35 min in rats (Lautt et al., 2001); the response in humans to a 50 mU/kg bolus reaches a peak at 27 min and is terminated by 88 min (Patarrao et al., 2007). The RIST can be carried out numerous times in the same animal preparation and has very high reproducibility. The RIST is not affected by pentobarbital anesthesia in rats (Latour and Lautt, 2002) nor isoflurane anesthesia in mice (Latour and Chan, 2002). It can be carried out in conscious mobile or anesthetized rats (Latour and Lautt, 2002; Sadri et al., 2006). The RIST index is able to be manipulated positively and negatively by pharmacological agents with sufficient sensitivity to produce dose-response curves (Takayama et al., 2000; Lautt et al., 2001).

A RIST carried out in recently fed animals demonstrates insulin action that can be compartmentalized as HISS-dependent and HISS-independent insulin action. The HISS-independent insulin action can be quantified by eliminating HISS release/action, for example by interfering with hepatic parasympathetic neurotransmission using atropine (Lautt et al., 2001). The use of atropine is validated by the lack of effect of atropine if HISS action has already been blocked by fasting or, in fed animals, by the observation that atropine completely reverses meal-induced insulin sensitization to result in a RIST index similar to that seen before the meal (Sadri et al., 2006).

Meal-induced insulin sensitization is not an artifact dependent upon the elevated postprandial levels of insulin and glucose as can be seen in the present study where the 2 h postprandial levels of insulin and glucose increased with age whereas the HISS component of insulin action progressively decreased. Further, atropine did not alter postprandial insulin or glucose levels (Afonso et al., 2007a; Xie and Lautt, 1995), yet atropine blocked HISS action and meal-induced insulin sensitization (Sadri et al., 2006). Thus, the degree of sensitization to insulin in response to a meal is mediated by what we refer to as HISS-dependent insulin action.

HISS is an unidentified blood-borne substance that is released from the liver in response to insulin and acts selectively on skeletal muscle (Xie and Lautt, 1996a; Moore et al., 2002). Although HISS action has been shown to be selective for skeletal muscle, the contribution of various tissues, such as liver, fat and brain, on the HISS-independent component is less clear. Nevertheless, the HISS-independent component is expected to largely represent responses of liver and adipose tissue. The liver and splanchnic organs appear to be insensitive to HISS action (Xie and Lautt, 1996a).

Hypothesis 1

HDIR accounts for insulin resistance that occurs with aging:

Ribeiro et al. (in press) first identified that insulin resistance that occurs with aging in Wistar rats is attributable entirely to a progressive development of HISS-dependent insulin resistance (HDIR). The HISS-independent component of insulin action was not affected.

The RIST index dramatically decreased (Fig. 4) from 174.6 ± 7.3 to 100.3 ± 6.1 by 6 months of age and further to 79.1 ± 4.0 mg kg−1 by 12 months in Sprague Dawley rats. The HISS-independent component of insulin action, quantified after atropine administration, was reduced significantly but to a lesser extent (RIST index was 80.3 ± 2.5 for 9 week rats and 64.8 ± 1.5 mg kg−1 for 52 week rats). Thus HDIR develops with age to the extent that by 1 year of age both Wistar and Sprague Dawley rats show greatly reduced HISS action and minor or insignificant impairment of the HISS-independent component of insulin action (Fig. 4).

Hypothesis 2

HDIR leads to increased adiposity in general without regional preferential impact:

To assess the impact on adiposity, we estimated total body fat mass and percent fat mass based on the electrical impedance method (Hall et al., 1989). In addition, fat mass was assessed from the weight of fat from three identifiable locations. Epididymal fat and perirenal fat do not drain into the splanchnic portal vein whereas perienteric fat mass drains directly into the portal vein and thus to the liver.

These data (Fig. 9), expressed either as total fat or percent of total body mass for each compartment, show no suggestion for selective roles for regional adiposity. Total fat mass in all areas increased dramatically by 6 months and further by 12 months. To test the hypothesis that the degree of HISS action is negatively associated with the degree of adiposity, correlation analysis was carried out. Fig. 7 shows the correlation of HISS-dependent insulin action with adiposity expressed as a percentage of whole body fat content or as weighed fat pad mass expressed as a percentage of body mass. Rats with normal high levels of HISS action show low adiposity. Adiposity progressively increases with the decline in HISS action (r2 = 0.75). The relationship is similar if the data are expressed based upon the combined or individual weighed fat depots.

Fig. 7
The effect of HISS-dependent insulin action on% whole body fat mass (FM) and total fat pad mass (% of body weight) in rats with normal diet or diet containing antioxidants.

Rats made obese by a high fat diet also showed a high correlation with HISS action and each fat pad, with the perinephric fat pad showing the most sensitive index (r2 = 0.87); perienteric (r2 = 0.67), and epididymal (r2 = 0.73). The high fat diet resulted in greatly accelerated HDIR and adiposity with all adipose indexes, as used here, showing no regional differentiation (Afonso et al., 2007a). Similar to the present study, the high fat diet resulted in insulin resistance almost entirely accounted for by HDIR. In both models HDIR is strongly associated with adiposity regardless of the specific index of adiposity chosen. As pertains to the view that intraperitoneal adiposity is more related to diabetes, the ratio of total fat mass to weighed fat mass does not change significantly as the total adiposity increases dramatically in these studies. The total weighed fat mass accounts for approximately one-third of the total body fat mass under all tested conditions. Therefore, no selective role for major regional adiposities can be supported from these data.

Indices of adiposity and the terminology used are highly variable in the literature. Fig. 2 shows the high correlation of whole body fat mass and total weighed fat pads either expressed as percentage of body weight (r2 = 0.87) or in absolute terms (r2 = 0.94). These data strongly validate the electrical impedance method as presented by Hall et al. (1989). However this method is sensitive to the placement of electrodes. The perienteric fat mass and the perinephric fat mass are excellent surrogate indicators of total body fat mass. The disadvantage with the perienteric fat mass measurements is the variability imposed by the nature of the peritoneal cavity where it is often difficult to separate fat from non-fat omentum. The perinephric fat mass is the easiest fat mass to dissect. The perinephric fat pad tended to increase to the greatest extent and the antioxidants conferred the greatest protection at that site. The simple weighing of the perinephric fat mass may be a reasonable surrogate for general adiposity. Although these data support the conclusion that the perinephric fat mass is an excellent indicator of overall adiposity, this relationship has only been evaluated for the high fat diet study (Afonso et al., 2007a) and the present study, and is contrary to the view that diabetes is related to specific regional adiposities. As frank diabetes was not attained, it remains possible that later stages of the progression of insulin resistance may show a regional effect on adiposity. Therefore, it is suggested that more than one index should be used until the issue of regional adiposity is further evaluated in other conditions. The two most straightforward regions of adiposity to further test the hypothesis of regional specialization would be the perienteric fat which drains into the portal vein and the perinephric fat which does not.

Hypothesis 3

HDIR represents a prediabetic state that progresses to diabetes and the metabolic syndrome:

In these studies the degree of HDIR and the duration of the experiments did not allow for a conclusive evaluation of the hypothesis. For purposes of discussion, HDIR is assumed to exist if the HISS-dependent component of insulin action is less than normal (≈55%) (e.g. Lautt et al., 2001). The fed young control rats in this cohort had HISS action of 52.3 ± 2.1%. HDIR progresses as the rats age and is associated with an increase in blood pressure and is strongly associated with a delayed or cumulative increase in adiposity. Postprandial insulin and glucose levels increased as HISS action decreased. The data are consistent with the conclusion that HDIR represents the prediabetic phase of insulin resistance resulting in a lipogenic state that progresses to full-blown diabetes and syndrome X. However, the changes in the syndrome X features and postprandial insulin and glucose (Table 1) were sufficiently small that this conclusion must be regarded as very tentative. To test this hypothesis fully, a more severe HDIR should be induced earlier on in the aging process and more time must be allowed for the impact of HDIR to develop. A limitation of this study is that HDIR at each age group is measured at that one time point, however, adiposity represents a lifetime accumulation and, therefore, will show a delayed effect.

4.2. Adiposity and HDIR – cause or effect

An argument against adiposity causing selective HDIR is based on the observation that the fatty Zucker rats (Afonso et al., 2007b) became obese but both the HISS-dependent and HISS-independent components of insulin action were equally affected there-by demonstrating that, at least in this leptin-deficient model of obesity, adiposity did not result in a selective impairment of the HISS action.

The HISS hypothesis predicts that HDIR results in a lipogenic prediabetic state and, therefore, HDIR should predict the level of adiposity (with some time delay as fat progressively accumulates). Fig. 7 shows that the relationship of HISS action to adiposity (slope of the plot) is strong and remains unaltered by the vitamin treatment. The absence of effect on this relationship suggests that any impact the antioxidant cocktail had on adiposity was due to protected HISS action and not an indirect effect whereby adiposity was attenuated by some other mechanism.

The time delay between HDIR and accumulated adiposity is clearly seen by the observation that HISS action had decreased by 66% in the control and 37% in the supplemented group at 6 months but no significant difference in adiposity was seen. If adiposity caused HDIR, the degree of HDIR should have been similar in the two groups. The difference in HDIR would be expected to result in a gradual delayed accumulation of fat that would only become significant after a period of time. By 12 months, HDIR had progressed and HISS action was reduced to only 17.0 ± 2.7% of the total response to the insulin bolus; the supplement conferred 60% protection of HISS action and 19% protection against adiposity. It is anticipated that had the protocol been extended to older ages, HDIR would have become complete and adiposity would have continued to preferentially occur in the untreated group. If adiposity had caused HDIR, there should be no time delay and both the treated and untreated groups at 6 months of age should have had equal reduction in HISS action.

Hypothesis 4

A targeted antioxidant cocktail can attenuate HIDR associated with age:

The dramatic decrease in RIST index seen at 6 months and 12 months was significantly attenuated by the antioxidant cocktail, Samec (Fig. 5). In addition, the attenuation can be attributed to the preservation of the HISS-dependent component of insulin action. Samec produced a minor but insignificant tendency to also protect the HISS-independent component. A limitation of this study is that the mechanism of the supplements to protect HISS action is unknown. Although the cocktail was formulated to quench free radicals in all cellular regions, the actual mechanism could be related to other effects of the cocktail especially considering the complexity of biochemistry affected by S-adenosylmethionine (Friedel et al., 1989; Mato et al., 2002).

There was a progressive increase in several parameters associated with syndrome X and these were attenuated by Samec. Postprandial glucose levels were elevated by 12 months (Table 1) and were protected by Samec so that the 12 month levels in the treated rats were not significantly different from the levels in the young rats. Samec did not significantly alter the slope or intercept of the relationship between HISS action and whole body fat mass, consistent with the hypothesis that Samec acted on adiposity by protecting HISS action.

The Samec supplement attenuated age-dependent dysfunctions including HDIR and parameters related to the metabolic syndrome. A crude but illustrative protection index can be calculated from the data in Table 1. The difference between the mean of a parameter determined for the young control group and the 12 month untreated group provides a quantitative estimate of the degree of dysfunction at that age. The difference between that parameter determined in the untreated and treated group represents the degree of protection afforded by Samec and is expressed as percent protection. The mild degree of hyperglycemia was attenuated in the one year group with 74% protection against 2 h postprandial glucose. The postprandial insulin levels were protected by 28%. The fat mass was protected by 19% and systolic blood pressure was protected by 31%. The dynamic insulin action determined in the fed state was protected by 29%. HISS action was reduced from 95 (young rats) to 15 mg kg−1 at 12 months but only to 40 mg kg−1 at 12 months in the supplement group thus representing a protection of 60%. The HISS-independent component of insulin action was decreased from 80 (young rats) to 65 mg kg−1 in untreated rats at 1 year and was also protected (33%) by the Samec treatment. In these animals, the antioxidant attenuation of HDIR offered protection against generalized adiposity, hypertension, and postprandial hyperglycemia.

In conclusion, dynamic insulin resistance occurred progressively with age and was accounted for by reduced HISS action, resulting in progressive HDIR. HDIR was strongly associated with adiposity as assessed from regional fat depots or whole body adiposity estimated from the bioelectrical impedance method. HDIR is a lipogenic state that results in generalized accumulation of fat. No regional fat zones appeared to have any selective relationship to HDIR. The unique antioxidant cocktail, consisting of vitamin C, E, and S-adenosylmethionine, confers protection against age-dependent HDIR, postprandial hyperglycemia and hyperinsulinemia, general adiposity, and hypertension. This suggests a potential approach for prevention of the early metabolic defect referred to as HDIR with resultant long-term impact on other parameters that HDIR leads to. The data suggest that the dysfunctions attenuated by treating HDIR may encompass the spectrum of deficiencies referred to variously as syndrome X, metabolic syndrome, insulin resistance syndrome, pre-diabetes, dysmetabolic syndrome, plurimetabolic syndrome, cardiometabolic syndrome, dyslipidemia, hypertension, hypertriglyceridemic waist, and deadly quartet (Grundy et al., 2005). Recently the American Diabetes Association (ADA) has suggested a new name “cardiometabolic risk” (Stern and Izkhakov, 2006). If, indeed, HDIR represents the prediabetic stage of insulin resistance that accounts for the progression to full stage diabetes and syndrome X, the associated risk factors should benefit from efforts directed to early detection of HDIR and abnormal meal-induced insulin sensitization, and to therapy directed toward the prevention or reversal of HDIR.


This study was funded by operating grants from the Canadian Institutes of Health Research and the Manitoba Health Research Council Regional Partnership Program. Manuscript preparation was by Karen Sanders. We acknowledge the excellent daily and long term care and monitoring of the animals provided by Gerry Nolette and the staff of Central Animal Care Services at the University of Manitoba.


Related Interests: Lautt and Ming are inventors on a patent for the synergistic combination of S-adenosylmethionine, vitamin E, and vitamin C (Samec) which is licensed to DiaMedica Inc., Winnipeg, Manitoba, Canada.


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