Modifications of citric acid cycle activity and gluconeogenesis in streptozotocin-induced diabetes and effects of metformin.

To better define the modifications of liver gluconeogenesis and citric acid cycle, or Krebs' cycle, activity induced by insulin deficiency and the effects of metformin on these abnormalities, we infused livers isolated from postabsorptive or starved normal and streptozotocin-induced diabetic rats with pyruvate and lactate (labeled with [3-13C]lactate) with or without the simultaneous infusion of metformin. Lactate and pyruvate uptake and glucose production were calculated. The 13C-labeling pattern of liver glutamate was used to calculate, according to Magnusson's model, the relative fluxes through Krebs' cycle and gluconeogenesis. These relative fluxes were converted into absolute values using substrate balances. In normal rats, starvation increased gluconeogenesis, the flux through pyruvate carboxylase-phosphoenolpyruvate carboxykinase (PC-PEPCK), and the ratio of PC to pyruvate dehydrogenase (PDH) flux (P < 0.05); metformin induced only a moderate decrease in the PC:PDH ratio. Livers from postabsorptive diabetic rats had increased lactate and pyruvate uptakes (P < 0.05); their metabolic fluxes resembled those of starved control livers, with increased gluconeogenesis and flux through PC-PEPCK. Starvation induced no further modifications in the diabetic group. Metformin decreased glucose output from the liver of starved diabetic rats (P < 0.05). The flux through PC-PEPCK and also pyruvate kinase were decreased (P < 0.05) by metformin in both groups of diabetic rats. In conclusion, insulin deficiency increased in this model of diabetes gluconeogenesis through enhanced uptake of substrate and increased flux through PC-PEPCK; metformin decreased glucose production by reducing the flux through PC-PEPCK.

Regulation of insulin receptor mRNA splicing in rat tissues. Effect of fasting, aging, and diabetes.

Recent findings suggested that alterations in insulin receptor isoform expression might be involved in the molecular mechanism of insulin resistance. Using reverse transcription reaction followed by competitive polymerase chain reaction, we measured the level of the receptor mRNA variants in rat insulin-sensitive tissues, under conditions of decreased insulin effectiveness (fasting, aging, and diabetes). The liver expressed the mRNA variant with exon 11 predominantly, and the hind limb skeletal muscles expressed the mRNA without exon 11. The heart and epididymal adipose tissue expressed both variants. Fasting and streptozocin-induced diabetes increased the level of receptor mRNAs in the liver but did not modify the repartition between the two variants. The modification of the expression ratio, in favor of the form with exon 11, found by some authors in the skeletal muscle of insulin-resistant patients was not observed in rat muscles that expressed > 99% of the form without exon 11 under all the conditions tested. In adipose tissue, the proportion of both mRNA variants was never altered (45% of exon 11-positive [Ex11+]), while the total receptor mRNA concentration changed markedly during fasting or aging. The only modification observed in the isoform distribution was a significant decrease in Ex11+ mRNA concentration in the liver, muscle, and heart of old rats. We conclude that alternative splicing of insulin receptor mRNA is not involved in the impairment of insulin action during fasting or diabetes. Its potential role in the insulin resistance of old animals remains to be defined.

In vivo expression of carbohydrate responsive element binding protein in lean and obese rats.

ChREBP (Carbohydrate response element binding protein) is considered to mediate the stimulatory effect of glucose on the expression of lipogenic genes. Its activity is stimulated by glucose. Less is known on the control of its expression. This expression could be controlled by nutritional (glucose, fatty acids) and hormonal (insulin) factors. We examined the in vivo nutritional control of ChREBP expression in liver and adipose tissue of Wistar rats. Compared respectively to the fed state and to a high carbohydrate diet, ChREBP mRNA concentrations were not modified by fasting or a high fat diet in rat liver and adipose tissue. FAS and ACC1 mRNA concentrations were on the contrary decreased as expected by fasting and high fat diets and these variations of FAS and ACC1 mRNA were positively related to those of SREBP-1c mRNA and protein, but not of ChREBP mRNA. Therefore i) ChREBP expression appears poorly responsive to modifications of nutritional condition, ii) modifications of the expression of ChREBP do not seem implicated in the physiological control of lipogenesis. To investigate the possible role of ChREBP in pathological situations we measured its mRNA concentrations in the liver and adipose tissue of obese Zucker rats. ChREBP expression was increased in the liver but not the adipose tissue of obese rats compared to their lean littermates. These results support a role of ChREBP in the development of hepatic steatosis and hypertriglyceridemia but not of obesity in this experimental model.

Ultrasonographic determination of residual urine in diabetic subjects: relationship to neuropathy and urinary tract infection.

The presence of residual urine was determined by postvoid bladder ultrasonography in 37 control subjects and 102 unselected insulin-dependent and non-insulin-dependent diabetic patients. Significant residual volume was detected in 19 diabetic subjects without explanation other than diabetic neurogenic bladder dysfunction in 15. The four others had prostatic hypertrophy. Excluding these four subjects, those with residual urine were slightly older than the others (P less than 0.05), but the known duration of diabetes was increased only for the insulin-dependent group (P less than 0.05). The presence of residual urine was strongly associated with peripheral neuropathy (P less than 0.001). In contrast, of all the manifestations of autonomic failure studied, only impotence (P less than 0.01) and decreased pupil motility (P less than 0.05) were associated with residual urine. The prevalence of urinary tract infection was higher in women than in men (P less than 0.02). This prevalence was increased in the presence of residual urine (P less than 0.02) but only for men (P less than 0.001).

Hypersensitivity to insulin during remissions in cyclosporin-treated IDDM patients.

OBJECTIVE: To test the sensitivity to insulin in recent-onset IDDM patients, its course according to treatment, and the advent of remissions. RESEARCH DESIGN AND METHODS: The euglycemic hyperinsulinemic clamp was used in 54 recent-onset IDDM patients and 14 healthy control subjects. Patients were tested after 1,2, and 4 wk of treatment with either insulin or insulin plus cyclosporin A, during cyclosporin A-associated long-lasting remissions, and during relapses. RESULTS: Insulin sensitivity was markedly decreased in all patients at onset. It was rapidly restored by insulin therapy, whether immunosuppression was associated with it or not. Insulin sensitivity was even higher than normal in the remission patients, who also were characterized by the reappearance of some endogenous insulin secretion and the sustained normalization of blood glucose profiles. During relapses, the deterioration of the blood glucose profiles was associated with some loss of insulin sensitivity. CONCLUSIONS: Cyclosporin A-associated remissions represent an original situation that associates euglycemia with the persistence of low endogenous insulin secretion. Cyclosporin A by itself had no influence on sensitivity to insulin, but allowed the reappearance of some insulin secretory capacity that contributed, with the improvement of insulin sensitivity, to the development of the diabetes honeymoon. The secretion of endogenous insulin, although lower than normal, was sufficient to secure a high sensitivity to insulin and the maintenance of normal blood glucose profiles, presumably because of the fact that insulin was released directly into the portal vein in these conditions. This metabolic state was precarious: the optimal sensitivity to insulin disappeared in patients who relapsed. These results have important clinical consequences: the preservation of islet residual secretory capacity by the use of newer nontoxic immunosuppressive protocols, combined with a minimal supportive insulin therapy in remission patients, may prolong remissions and maintain an optimal insulin sensitivity.

Role of catecholamines in the ketonemic response to somatostatin in normal man.

In order to determine if a rise of circulating catecholamines occurs during somatostatin (SRIF) infusion in normal man, and if this increase plays a significant metabolic role, we infused four normal subjects with SRIF (500 micrograms/h) alone or associated with either alpha-(phentolamine) or beta-(propranolol) adrenergic blocking agents. During SRIF infusion, the initial small decrease in blood glucose was followed by a rise of epinephrine from 25-46 ng/liter (range) to 117-143 ng/liter (range) (P less than 0.05) at 80 min and norepinephrine from 204 +/- 16 to 418 +/- 60 ng/liter at 90 min (P less than 0.05). Thereafter, plasma nonesterified fatty acids, blood glycerol, and ketone bodies increased significantly. Phentolamine adjunction modified neither the catecholamines rise, nor the metabolic changes. Propranolol adjunction did not modify the glucose fall and the catecholamine rise, but resulted in blunted increments of fatty acids and glycerol and in an almost complete suppression of the increase of ketone bodies. These results suggest that the enhanced lipolysis and ketogenesis observed during SRIF infusion are not only due to the SRIF-induced insulin deficiency but also in part to a beta-receptor mediated effect of catecholamines.

Effects of ketone bodies on basal and insulin-stimulated glucose utilization in man.

Using the euglycemic clamp technique, we investigated the effects of high ketone body levels on basal and insulin-stimulated glucose utilization in normal subjects. Infusion of sodium acetoacetate in the postabsorptive state raised ketone body levels from 150 +/- 20 (+/- SE) mumol/liter to more than 1 mmol/liter. Endogenous glucose production declined from 2.71 +/- 0.20 mg kg-1 min-1 to 1.75 + 0.26 (P less than 0.01) and glucose utilization from 2.71 +/- 0.20 to 1.98 +/- 0.17 mg kg-1 min-1 (P less than 0.01), while blood glucose was maintained at the initial level by the infusion of glucose. There were no changes in plasma glucagon, insulin, or C-peptide. Plasma nonesterified fatty acids (P less than 0.01) and blood glycerol (P less than 0.01) and alanine (P less than 0.05) decreased, while blood lactate increased (P less than 0.01). Infusion of sodium bicarbonate had no effect on glucose kinetics. The decreases in glucose utilization and endogenous glucose production during the infusion of acetoacetate were not modified when the fall of plasma nonesterified fatty acids was prevented by iv heparin injection. During control euglycemic hyperinsulinemic clamps (1 and 10 mU kg-1 min-1 insulin infusion), endogenous glucose production was suppressed at the lowest insulin infusion rate; glucose utilization increased first to 7.32 +/- 0.96 mg kg-1 min-1 and then to 16.5 +/- 1.27 mg kg-1 min-1. During euglycemic hyperinsulinemic clamps with simultaneous sodium acetoacetate infusion, similar insulin levels were attained; endogenous glucose production was also suppressed at the lowest insulin infusion rate, and insulin-stimulated glucose utilization rates (7.93 +/- 1.70 and 15.80 +/- 1.30 mg kg-1 min-1) were not modified. In conclusion, acetoacetate infusion decreased basal, but not insulin-stimulated, glucose utilization. The increase in lactate during acetoacetate infusion in the postabsorptive state suggests that ketone body acted by decreasing pyruvate oxidation.

Lipolytic and ketogenic fluxes in human hyperthyroidism.

The effect of hyperthyroidism on lipolytic and ketogenic fluxes was determined by measuring simultaneously (stable isotope methodology) glycerol, nonesterified fatty acids (NEFA), and ketone body (KB) kinetics in euthyroid and hyperthyroid subjects. In the postabsorptive state hyperthyroid patients had normal concentrations of insulin and glucagon, but increased concentrations (P less than 0.01) and turnover rates (P less than 0.01) of glycerol, NEFA, and KB. The ratio of NEFA appearance rate to glycerol appearance rate was decreased in hyperthyroid subjects (2.34 +/- 0.23 vs. 3.15 +/- 0.22; P less than 0.05), indicating that intracellular cycling between triglycerides and fatty acids was increased. The percentage of NEFA flux used for KB production, calculated from NEFA disappearance rates and KB appearance rates, was increased in hyperthyroid patients (21.20 +/- 2.75% vs. 13.37 +/- 0.63%; P less than 0.05), suggesting a diversion during hyperthyroidism of hepatic fatty acid metabolism toward ketogenesis. However, when the plasma NEFA levels of control subjects were raised by the infusion of a triglyceride emulsion to levels comparable to those observed in hyperthyroid patients their percentage of NEFA flux used for ketogenesis rose to values slightly higher (26.30%) than those of hyperthyroid subjects. In conclusion, 1) hyperthyroidism results not only in increased lipolysis, but also in enhanced triglyceride-fatty acid cycling, which could contribute to the excessive energy expenditure; and 2) the increased KB production of hyperthyroid patients results more from an increase in NEFA availability than from a direct stimulation of hepatic ketogenesis.

Hepatic lipogenesis and cholesterol synthesis in hyperthyroid patients.

To determine the effect of hyperthyroidism on hepatic lipogenesis and cholesterol synthesis we measured these metabolic pathways (deuterated water method) in euthyroid and hyperthyroid subjects investigated in the postabsorptive state. Hyperthyroid patients had increased concentrations of glucose (P < 0.05), insulin (P < 0.05), nonesterified fatty acids (P < 0.01), and triglycerides (P < 0.05) and decreased levels of plasma cholesterol (P < 0.01). The contribution of hepatic lipogenesis to plasma triglycerides was largely increased in hyperthyroid subjects (23.0 +/- 1.8% vs. 7.5 +/- 0.2%; P < 0.001), whereas the fractional synthetic rate of cholesterol was moderately higher (5.0 +/- 0.8% vs. 3.3 +/- 0.2%; P < 0.05). mRNA levels of beta-hydroxy-beta-methyl glutaryl-coenzyme A reductase, measured in circulating mononuclear cells, were increased (P < 0.05), whereas those of low density lipoprotein (LDL) receptor and LDL receptor-related protein were unchanged. Sterol responsive element binding protein-1c mRNAs were undetectable in mononuclear cells from both groups of subjects. The large stimulation of hepatic lipogenesis in hyperthyroid patients is probably explained by both a direct action of thyroid hormones and the increase in insulin. It could contribute to their moderate rise in triglycerides levels. The decreased plasma cholesterol level is observed despite an enhanced synthetic rate and is thus related to an increased clearance rate. The lack of increased expression of LDL receptor and LDL receptor-related protein suggests that other receptors are implicated.

Glucose metabolism in experimental hyperthyroidism: intact in vivo sensitivity to insulin with abnormal binding and increased glucose turnover.

The characteristics of the dose response of insulin on the glucose turnover rate and erythrocyte insulin binding parameters were determined in five normal men before and during experimentally induced hyperthyroidism [L-T4 (2 micrograms kg-1 day-1) for 4 weeks with additional L-T3 (1 microgram kg-1 day-1) for the following 3 weeks]. Hyperthyroidism was characterized by significant rises in T3 from 1.92 +/- 0.17 (+/- SEM) to 3.66 +/- 0.17 nmol/liter (P less than 0.01) and resting metabolic rate from 39 +/- 0.7 to 48 +/- 1 watt/m2 (P less than 0.001). While the subjects received a diet adapted to the metabolic rate, blood glucose rose from 3.8 +/- 0.07 to 4.46 +/- 0.11 mmol/liter (P less than 0.05) without a significant change in plasma insulin. During the insulin dose-response study, glucose infusion rates were unaltered by hyperthyroidism, and neither the maximum effect nor the sensitivity to insulin was altered. Glucose turnover rate, measured using [6,6-2H2]glucose as tracer, was determined in the basal state and during the 0.4 mU kg-1 min-1 insulin infusion. In the basal state, it was significantly increased by hyperthyroidism (control, 2.3 +/- 0.1; hyperthyroidism, 3.7 +/- 0.1 mg kg-1 min-1). During the insulin infusion, hepatic glucose production was totally suppressed before T4 and T3 treatment, but was 0.96 +/- 0.39 mg kg-1 min-1 during T4 and T3 treatment. A marked decrease in the insulin binding affinity to erythrocytes was found without a change in the insulin receptor number. In conclusion, glucose metabolism in experimental hyperthyroidism is characterized by 1) increases in basal glucose production and utilization; 2) antagonism between the effect of insulin and hyperthyroidism at the hepatic level; and 3) lack of peripheral insulin resistance in spite of marked alteration in erythrocyte insulin binding affinity.

Effect of epinephrine on the relationship between nonesterified fatty acid availability and ketone body production in postabsorptive man: evidence for a hepatic antiketogenic effect of epinephrine.

The effect of epinephrine (EPI) on the transformation of nonesterified fatty acids (NEFA) into ketone bodies (KB) in normal subjects was determined by measuring simultaneously NEFA ([1-13C]palmitic acid) and KB ([3-13C]- or [3,4-13C2]acetoacetate) kinetics at different NEFA levels in the presence of basal (control test) or increased (EPI infusion test) EPI concentrations. During the control test the initial (postabsorptive state) concentrations and turnover rates of NEFA and KB were 476 +/- 47 (+/- SEM) and 4.30 +/- 0.17 mumol kg-1 min-1 (NEFA) and 126 +/- 17 and 2.49 +/- 0.07 mumol kg-1 min-1 (KB). The fraction of NEFA converted into KB was between 11.5-14.6%. Raising NEFA levels to about 650 mumol L-1 (iv infusion of a triglyceride emulsion) resulted in an increase in this fraction to between 26-30.3% (P less than 0.01). When NEFA concentrations were next abruptly raised to high levels (near 3 mmol L-1) by heparin injection this fraction returned to near the initial values (15-19.2%). During the EPI infusion test the initial (postabsorptive) concentrations and turnover rates of NEFA and KB as well as the fraction of NEFA converted into KB (10.5-11.5%) were comparable to the initial values of the control test. Intravenous infusion of EPI (10 ng kg-1 min-1) raised NEFA between 600 and 750 mumol L-1, comparable to values during the triglyceride test, but the fraction of NEFA converted into KB remained between 8.2-12% (P less than 0.05 vs. control test); when NEFA then were raised to even higher values (near 2.5 mmol L-1) by the infusion of a triglyceride emulsion and the injection of heparin, this fraction decreased to between 4-8% (P less than 0.05 vs. initial values of the EPI test and P less than 0.05 vs. the control test). In conclusion, 1) the fraction of NEFA converted into KB appears to depend in part on the NEFA concentration; and 2) the net effect of EPI infusion was to decrease the fraction of NEFA converted into KB.

Hormonal and metabolic effects of near physiological increase of plasma immunoreactive somatostatin 14.

The possibility that somatostatin 14 (SRIF) may exert true endocrine actions in man was tested by investigating the hormonal and metabolic effects of the peptide infused for 80 min at rates of 36.5, 73, and 146 pmol kg-1 h-1 in six healthy subjects who fasted overnight. These three doses increased the level of plasma SRIF-like immunoreactivity in the range of concentrations recorded postprandially with the same assay system. These low SRIF infusion rates decreased insulinemia and to a lesser extent glucagonemia, and increased glucosemia and ketonemia. Both the reduction of insulin and the increase of glucosemia were significantly related to the increase of plasma SRIF-like immunoreactivity. All parameters returned to control values upon discontinuing the peptide infusion. This study suggests that SRIF may have an endocrine role in man and that such low dose, short time SRIF infusions could exert metabolic effects different from those of larger, probably pharmacological, infusion rates.

Effects of isoenergetic high-carbohydrate compared with high-fat diets on human cholesterol synthesis and expression of key regulatory genes of cholesterol metabolism.

BACKGROUND: High-carbohydrate diets improve plasma cholesterol concentrations but increase triacylglycerol concentrations; the latter effect increases the risk of cardiovascular disease (CVD). Triacylglycerol concentrations increase only during very-high-carbohydrate diets consisting mainly of simple sugars. OBJECTIVE: We compared the CVD risk profile, cholesterol metabolism, and glucose tolerance of 7 healthy subjects during 2 isoenergetic diets: a high-fat, low-carbohydrate diet (HF diet) and a moderately high-carbohydrate, low-fat diet (HC diet). DESIGN: In a randomized crossover study, we measured the effects of the HF diet [40% carbohydrate and 45% fat (15% saturated, 15% monounsaturated, and 15% polyunsaturated)] and HC diet [55% carbohydrate (mainly complex) and 30% fat (10% saturated, 10% monounsaturated, and 10% polyunsaturated)] (3 wk each) on plasma lipid concentrations, oral glucose tolerance, cholesterol synthesis rate, and the messenger RNA (mRNA) concentrations of beta-hydroxy-beta-methylglutaryl coenzyme A (HMG-CoA) reductase, the LDL receptor, and the LDL-receptor-related protein (LRP). RESULTS: Compared with the HF diet, the HC diet lowered total, LDL, and HDL cholesterol (P < 0.05 for all) without modifying the ratio of LDL to HDL cholesterol; triacylglycerol concentrations were unchanged. Lower cholesterol concentrations occurred despite a higher cholesterol synthesis rate (P < 0.05) and higher HMG-CoA reductase mRNA concentrations (P < 0.05). LDL receptor mRNA concentrations were unchanged, LRP mRNA concentrations were lower (P < 0.01), and oral glucose tolerance was better (P < 0.05) with the HC diet. CONCLUSION: The beneficial effects of the HC diet on glucose tolerance and plasma cholesterol concentrations without increases in triacylglycerol show that this diet had favorable effects on both insulin sensitivity and the plasma lipid profile.

Decreased glucose-induced thermogenesis at the onset of obesity.

To investigate the possible existence of a defect of thermogenesis at the onset of obesity, we studied glucose-induced thermogenesis (GIT) during an oral glucose-tolerance test (OGTT) (1 g/kg body wt) in 12 women who were at the onset of obesity (group A) compared with 12 long-standing obese women (group B) and 8 lean control subjects. During OGTT hyperinsulinemia and glucose intolerance were measured in group B, suggesting an insulin-resistant state, but not in group A. A similar defect in GIT occurred in both obese groups (8.9 +/- 1.5% in control subjects vs 4.2 +/- 1.1% in group A and 4.3 +/- 1.0% in group B, P < 0.05) despite the absence of alteration in nonoxidative glucose metabolism. We conclude that a decrease in GIT already exists at the onset of obesity, which supports the hypothesis of a possible involvement of this defect in the genesis of obesity.

Influence of human obesity on the metabolic fate of dietary long- and medium-chain triacylglycerols.

The metabolic fate of an oral long-chain-triacylglycerol (LCT) load and of a mixed oral LCT and medium-chain-triacylglycerol (MCT) load was followed for 6 h in eight control and eight obese subjects with normal postabsorptive triacylglycerol concentrations. Labeled triacylglycerol and indirect calorimetry were used. Results showed that LCTs were less oxidized in obese than in control subjects (3.2+/-0.5 compared with 6.0+/-0.4 g, P < 0.01). Moreover, the amount of LCT oxidized was negatively correlated with fat mass (r = -0.77, P < 0.01). Appearance in plasma of dietary triacyglycerol-derived long-chain fatty acids was blunted in obese subjects and it was negatively related to fat mass (r = -0.84, P < 0.01) and positively to LCT oxidation (r = 0.70, P < 0.01). On the contrary, MCT oxidation was not altered in obese subjects compared with control subjects. Furthermore, the proportion of MCTs oxidized was higher in both groups compared with LCTs (x+/-SEM: 57.5+/-2.6% compared with 15.2+/-1.6%, P < 0.01, n = 16). Our conclusion is that obesity is associated with a defect in the oxidation of dietary LCTs probably related to an excessive uptake by the adipose tissue of meal-derived long-chain fatty acids. MCTs, the oxidation of which is not altered in obesity, could therefore be of interest in the dietary treatment of obesity.

Hormonal control of glucose production and pyruvate kinase activity in isolated rat liver cells: influence of hypothyroidism.

Hormonal control of glucose production and of L-pyruvate kinase activity has been measured in isolated liver cells from fed control and thyroidectomized rats. In hypothyroid rats, sensitivity to isoproterenol as measured by these parameters was increased: the apparent K0.5 for isoproterenol-induced stimulation of glucose production decreased from 8.0 +/- 3 X 10(-6) M in control rats to 2.0 +/- 0.2 X 10(-8) M in hypothyroid rats (P less than 0.001) and the apparent K0.5 for inhibition of L-pyruvate kinase was 5 +/- 2 X 10(-7) M vs. 7 +/- 2 X 10(-9) M (P less than 0.001) in control and thyroidectomized rats, respectively. Utilisation of specific adrenergic antagonists confirmed increased beta-adrenergic responsiveness in hypothyroid rats. This phenomenon was not reversed by 3 days of T3 treatment (10 micrograms/100 g body weight). Sensitivity to the alpha-agonist was unchanged by thyroid status. Stimulation of glucose production and inhibition of L-pyruvate kinase activity by glucagon and their reversal by insulin were not affected by hypothyroidism. The dose-response curve to vasopressin and its maximal effect measured on stimulation of glucose production were unchanged in thyroidectomized rats. Thus, hypothyroidism produces a specific enhancement of liver beta-adrenergic responsiveness without affecting sensitivity to glucagon, insulin and vasopressin.

In vivo measurement of plasma cholesterol and fatty acid synthesis with deuterated water: determination of the average number of deuterium atoms incorporated.

Fractional lipid synthesis can be measured using the incorporation of deuterium from deuterated water. The calculations require knowledge of the maximum incorporation number (N) of deuterium atoms in the molecules synthesized. For both tissue palmitate and cholesterol, N values have been found to be higher during in vivo versus in vitro experiments. We determined the N values to be used for measuring the fractional synthesis of plasma cholesterol and of palmitate triglycerides (TG). Rats were given drinking water enriched (7% to 10%) with deuterated water, and N was determined from the mass isotopomer distributions of plasma cholesterol and plasma TG palmitate and the deuterium enrichment of plasma water. We found N to be 21 for palmitate and 27 for cholesterol. These values agree with those reported for tissue palmitate and cholesterol in vivo, and are higher than values found in vitro. We also found large deuterium enrichments in plasma glucose and in liver lactate and pyruvate. We suggest that, compared with in vitro studies, in vivo metabolism of these compounds leads to an additional pathway of incorporation of deuterium into lipids through deuterium-labeled acetyl coenzyme A (CoA). This could explain why N values are higher in vivo than in vitro.

Glucose production and gluconeogenesis in postabsorptive and starved normal and streptozotocin-diabetic rats.

Using a 3-hour primed-continuous infusion of [3-3H]glucose and [2-13C]glycerol, we measured glucose production, gluconeogenesis from glycerol, and total gluconeogenesis (using mass isotopomer distribution analysis [MIDA] of glucose) in postabsorptive and starved normal and streptozotocin-diabetic rats. In normal rats, 48 hours of starvation increased (P < .01) the percent contribution of both gluconeogenesis from glycerol (from 14.4% +/- 1.8% to 25.5% +/- 4.0%) and total gluconeogenesis (from 52.2% +/- 3.9% to 89.8% +/- 1.3%) to glucose production, but the absolute gluconeogenic fluxes were not modified, since glucose production decreased. Diabetic rats showed increased glucose production in the postabsorptive state; this decreased with starvation and was comparable to the of controls after 48 hours of starvation. Gluconeogenesis was increased in postabsorptive diabetic rats (69.0% +/- 1.3%, P < .05 v controls). Surprisingly, this contribution of gluconeogenesis to glucose production was not found to be increased in 24-hour starved diabetic rats (64.4% +/- 2.4%). These rats had significant liver glycogen stores, but gluconeogenesis was also low (42.8% +/- 2.1%) in 48-hour starved diabetic rats deprived of glycogen stores. Moreover, in 24-hour starved diabetic rats infused with [3-13C]lactate, gluconeogenesis was 100% when determined by comparing circulating glucose and liver pyruvate enrichment, but only 47% +/- 3% when calculated from the MIDA of glucose. Therefore, MIDA is not a valid method to measure gluconeogenesis in starved diabetic rats. This was not explained by differences in the labeling of liver and kidney triose phosphates: functional nephrectomy of starved diabetic rats decreased glucose production, but gluconeogenesis calculated by the MIDA method was only 48% +/- 3.3%. We conclude that (1) diabetic rats have increased glucose production and gluconeogenesis in the postabsorptive state; (2) starvation decreases glucose production and increases the contribution of gluconeogenesis, but MIDA is not an appropriate method in this situation; and (3) the kidneys contribute to glucose production in starved diabetic rats.

Effects of different lipid substrates on glucose metabolism in normal postabsorptive humans.

We investigated the effects on glucose metabolism of the infusion of either long-chain triglycerides (LCT), a mixture of long-chain and medium-chain triglycerides (MCT/LCT), D-beta-hydroxybutyrate (D-beta-OHB), or saline in normal postabsorptive subjects. Plasma insulin, C-peptide, and glucagon concentrations were unchanged in all groups. LCT and MCT/LCT infusions increased levels of plasma free fatty acids (FFA) compared with those of the saline group, whereas D-beta-OHB decreased them. Plasma ketone body concentrations were higher during the D-beta-OHB and triglyceride infusions than during the saline test. Glucose concentrations and appearance (Ra) and disappearance (Rd) rates were not modified during saline infusion. Glucose levels decreased only in the D-beta-OHB and MCT/LCT groups (P < .05), whereas they were unchanged during LCT infusion. Glucose Ra decreased slightly by 15% to 17% in LCT, MCT/LCT, and D-beta-OHB groups (P < .05 v saline). Glucose Rd decreased by 14% to 16% in each lipid-infusion group (P < .05 v saline). Glucose clearance rates decreased by 14% only in the LCT group (P < .001). Glucose oxidation rates did not change significantly during the lipid substrate infusions compared with saline infusion. In conclusion, (1) the effects of fatty acids on glucose metabolism appear to depend on the fatty acid chain length, since only LCT infusion significantly impaired glucose utilization; and (2) in subjects with normal endocrine pancreas function, we found no adverse effects of a short-term increase in lipid substrate availability on glucose production rate and concentration.

Measuring glycerol turnover, gluconeogenesis from glycerol, and total gluconeogenesis with [2-13C] glycerol: role of the infusion-sampling mode.

Mass isotopomer distribution analysis (MIDA) of glucose during infusion of [2-13C]glycerol is a new method for measuring total gluconeogenesis (GNG). Since this method relies on calculation of the isotopic enrichment (IE) of hepatic triose phosphates (TP), the results should be independent of the sites of tracer infusion and blood sampling. Postabsorptive and starved rats were infused with [2-13C]glycerol and sampled either in the arterial-venous (A-V) or venous-arterial (V-A) modes. Blood was also sampled from the portal vein. In both postabsorptive and starved rats, glycerol turnover rate (Rt) and the percent contribution of glycerol to total glucose production were higher in the A-V mode than in the V-A mode (P < .05). Glycerol IE in portal venous blood was intermediate between IE values observed in peripheral arterial and venous blood. Its use for calculating the contribution of glycerol to glucose production reconciled the results obtained with the two infusion-sampling modes in both postabsorptive and starved rats; this contribution was increased by starvation (P < .01). In postabsorptive rats, total GNG calculated from MIDA of glucose accounted for approximately 50% of glucose production whatever the infusion-sampling mode (A-V, 48.8% +/- 4.7%; V-A, 52.2% +/- 3.9%). This contribution increased to 90% in starved rats, again, with no difference between A-V (95.2% +/- 1.8%) and V-A (89.2% +/- 1.3%) modes. In conclusion, during infusion of [2-13C]glycerol, total GNG measured from MIDA of glucose is independent of the infusion-sampling mode, contrary to calculations of Rt and GNG from glycerol. Measurement of glycerol IE in portal venous blood reconciles the results obtained with the two modes with respect to the contribution of glycerol to GNG.