Interactions between insulin and thyroid hormone in the control of lipogenesis.

1. The effects of intragastric glucose feeding and L-tri-iodothyronine (T3) administration on rates of hepatic and brown-fat lipogenesis in vivo were examined in fed and 48 h-starved rats. 2. T3 treatment increased hepatic lipogenesis in the fed but not the starved animals. Brown-fat lipogenesis was unaffected or slightly decreased by T3 treatment of fed or starved rats. 3. Intragastric glucose feeding increased hepatic lipogenesis in control or T3-treated fed rats, but did not increase hepatic lipogenesis in starved control rats. Glucose feeding increased hepatic lipogenesis if the starved rats were treated with T3. Glucose feeding increased rates of brown-fat lipogenesis in all experimental groups. The effects of glucose feeding on liver and brown-fat lipogenesis were mimicked by insulin injection. 4. The increase in hepatic lipogenesis in T3-treated 48 h-starved rats after intragastric glucose feeding was prevented by short-term insulin deficiency, but not by (-)-hydroxycitrate, an inhibitor of ATP citrate lyase. The increase in lipogenesis in brown adipose tissue in response to glucose feeding was inhibited by both short-term insulin deficiency and (-)-hydroxycitrate. 5. The results tend to preclude pyruvate kinase and acetyl-CoA carboxylase as the sites of interaction of insulin and T3 in the regulation of hepatic lipogenesis in 48 h-starved rats. Other potential sites of interaction are discussed.     

Involvement of insulin receptor substrates in epidermal growth factor induced activation of phosphatidylinositol 3-kinase in rat hepatocyte primary culture.

Short-term incubation of adult rat hepatocytes with epidermal growth factor (EGF) caused tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and IRS-2 when the cells had been submitted to primary culture from 1-18 h. Tyrosine-phosphorylated IRS-1 and IRS-2 bound to the regulatory subunit (p85) of phosphatidylinositol (PtdIns) 3-kinase, thereby activating the enzymic activity. Tyrosine phosphorylation of the IRSs and activation of PtdIns 3-kinase in 3 h cultured hepatocytes both proceeded similarly to the same actions of insulin; the activation was rapid and transient, with peak values at 15-30 s and with similar EC(50)s in the nM range in both cases. A possible involvement of insulin receptors in these insulin-like actions of EGF was excluded by the following three lines of evidence. Insulin caused tyrosine phosphorylation of the insulin receptor beta-subunit but EGF did not. In contrast, the EGF receptor was phosphorylated by EGF, but the insulin receptor was not. The actions of EGF, but not those of insulin, were inhibited by AG1478, a selective inhibitor of EGF receptor tyrosine kinase. Cultured hepatocytes exposed to insulin or insulin-like growth factor-I (IGF-I) for a short period responded to the subsequent addition of EGF, whereas EGF-treated cells responded to insulin. The cells, however, displayed receptor desensitization under the same conditions, that is, no response was observed upon repeated addition of the same agonist, EGF, insulin or IGF-I. Thus, the EGF receptor-initiated signalling was mediated by PtdIns 3-kinase associated with tyrosine-phosphorylated IRSs in short-term cultured rat hepatocytes.     

Permissive action of triiodothyronine on the long-term increase of glycolysis by insulin in cultured rat hepatocytes

The long-term influence of triiodothyronine (T3) and insulin on glycolysis, some glycolytic/gluconeogenic enzymes and insulin responsiveness and sensitivity was investigated in rat hepatocytes cultured for 48 h without T3, with 10 microM T3, with 10nM insulin and with insulin plus T3. From 48 h-51 h basal glycolysis ([14C]lactate formation from [14C]glucose) was measured in the absence and short-term insulin-stimulated glycolysis in the presence of 100 nM insulin. 1) T3 addition for 48 h had no significant influence on basal or on insulin-stimulated glycolysis. 2) Insulin addition for 48 h increased basal glycolysis to 300%, and insulin-stimulated glycolysis to 160%. 3) T3 plus insulin addition for 48 h elevated basal glycolysis to 560% and insulin-stimulated glycolysis to 230%. 4) The 48-h treatment with T3 did not change glucokinase (GK) and pyruvate kinase (PK) activity, yet it increased phosphoenol-pyruvate carboxykinase (PEPCK) activity to 150%. 5) The 48-h treatment with insulin as well as T3 plus insulin enhanced GK to 200% and PK to 140% and decreased PEPCK to 65%. 6) The long-term effect of T3 on glycolysis was maximal at initial concentrations of 100 nM. 7) The long-term treatment with T3 did not alter the short-term responsiveness or sensitivity of glycolysis for insulin, neither in cells from euthyroid nor from hypothyroid rats. The present results allow the conclusion that T3 had a permissive effect on the long-term increase of glycolysis by insulin, and that T3 exerted this function by altering neither the cellular content of key enzymes nor the short-term insulin responsiveness and sensitivity.     

Metabolic Effects of CX3CR1 Deficiency in Diet-Induced Obese Mice

The fractalkine (CX3CL1-CX3CR1) chemokine system is associated with obesity-related inflammation and type 2 diabetes, but data on effects of Cx3cr1 deficiency on metabolic pathways is contradictory. We examined male C57BL/6 Cx3cr1^-/- mice on chow and high-fat diet to determine the metabolic effects of Cx3cr1 deficiency. We found no difference in body weight and fat content or feeding and energy expenditure between Cx3cr1^-/- and WT mice. Cx3cr1^-/- mice had reduced glucose intolerance assessed by intraperitoneal glucose tolerance tests at chow and high-fat fed states, though there was no difference in glucose-stimulated insulin values. Cx3cr1^-/- mice also had improved insulin sensitivity at hyperinsulinemic-euglycemic clamp, with higher glucose infusion rate, rate of disposal, and hepatic glucose production suppression compared to WT mice. Enhanced insulin signaling in response to acute intravenous insulin injection was demonstrated in Cx3cr1^-/- by increased liver protein levels of phosphorylated AKT and GSK3β proteins. There were no differences in adipose tissue macrophage populations, circulating inflammatory monocytes, adipokines, lipids, or inflammatory markers. In conclusion, we demonstrate a moderate and reproducible protective effect of Cx3cr1 deficiency on glucose intolerance and insulin resistance.     

Deficiency of the mitochondrial electron transport chain in muscle does not cause insulin resistance

Background

It has been proposed that muscle insulin resistance in type 2 diabetes is due to a selective decrease in the components of the mitochondrial electron transport chain and results from accumulation of toxic products of incomplete fat oxidation. The purpose of the present study was to test this hypothesis.

Methodology/Principal Findings

Rats were made severely iron deficient, by means of an iron-deficient diet. Iron deficiency results in decreases of the iron containing mitochondrial respiratory chain proteins without affecting the enzymes of the fatty acid oxidation pathway. Insulin resistance was induced by feeding iron-deficient and control rats a high fat diet. Skeletal muscle insulin resistance was evaluated by measuring glucose transport activity in soleus muscle strips. Mitochondrial proteins were measured by Western blot. Iron deficiency resulted in a decrease in expression of iron containing proteins of the mitochondrial respiratory chain in muscle. Citrate synthase, a non-iron containing citrate cycle enzyme, and long chain acyl-CoA dehydrogenase (LCAD), used as a marker for the fatty acid oxidation pathway, were unaffected by the iron deficiency. Oleate oxidation by muscle homogenates was increased by high fat feeding and decreased by iron deficiency despite high fat feeding. The high fat diet caused severe insulin resistance of muscle glucose transport. Iron deficiency completely protected against the high fat diet-induced muscle insulin resistance.

Conclusions/Significance

The results of the study argue against the hypothesis that a deficiency of the electron transport chain (ETC), and imbalance between the ETC and β-oxidation pathways, causes muscle insulin resistance.     

Effects of diabetes and insulin on ketone bodies metabolism in heart

This work investigates the effect of alloxan-induced short-term diabetes (24 h) on D-3-hydroxybutyrate metabolism at physiological and non-physiological concentrations of the ketone body in the isolated non-working perfused rat heart. Also the effect of insulin (2 mU.ml⁻¹) on D-3-hydroxybutyrate metabolism was investigated in hearts from normal and diabetic rats. The rates of D-3-hydroxybutyrate utilization and oxidation and of acetoacetate production were proportional to D-3-hydroxybutyrate concentration. The utilization of D-3-hydroxybutyrate showed saturation kinetics in hearts from normal and diabetic rats, in the presence and absence of insulin. Acute short-term diabetes augmented D-3-hydroxybutyrate utilization and oxidation at 1.25 and 2.5 mM DL-3-HB, with no significant effect at higher concentrations, but increased acetoacetate production at all investigated concentrations. In hearts from normal rats, insulin enhanced D-3-hydroxybutyrate utilization and oxidation at 2.5, 5, and 10 mM DL-3-HB, but no effect was observed at the lowest (1.25 mM) and highest (16 mM) DL-3-HB concentrations. Insulin had no effect on D-3-hydroxybutyrate metabolism in hearts from diabetic rats. No significant effect of insulin on the rate of acetoacetate production in normal and diabetic states was observed.     

Regulation of glycogen metabolism in primary cultures of rat hepatocytes. Restoration of acute effects of glucose in cells from diabetic rats involves protein synthesis

Defective acute regulation of hepatic glycogen synthase by glucose and insulin, caused by severe insulin deficiency, can be corrected in adult rat hepatocytes in primary culture by inclusion of insulin, triiodothyronine, and cortisol in a chemically defined serum-free culture medium over a 3-day period (Miller, T. B., Jr., Garnache, A. K., Cruz, J., McPherson, R. K., and Wolleben, C. (1986) J. Biol. Chem. 261, 785-790). Using primary cultures of hepatocytes isolated from normal and diabetic rats in the same serum-free chemically defined medium, the present study addresses the effects of cycloheximide and actinomycin D on the chronic actions of insulin, triiodothyronine, and cortisol to facilitate the direct effects of glucose on the short-term activation of glycogen synthase. The short-term presence (1 h) of the protein synthesis blockers had no effect on acute activation of glycogen synthase by glucose in primary hepatocyte cultures from normal rats. Normal cells maintained in the presence of cycloheximide or actinomycin D for 2 and 3 days exhibited unimpaired responsiveness to glucose activation of synthase. The protein synthesis inhibitors were effective at blocking the restoration of glucose activation of synthase in diabetic cells in media which restored the activation in their absence. Restoration of glycogen synthase phosphatase activity by insulin, triiodothyronine, and cortisol in primary cultures of diabetic hepatocytes was also blocked by cycloheximide or actinomycin D. These data clearly demonstrate that restoration of acute glycogen synthase activation by glucose and restoration of glycogen synthase phosphatase activity in primary cultures of hepatocytes from adult diabetic rats are dependent upon the synthesis of new protein.     

Differential regulation of amino acid transporter SNAT3 by insulin in hepatocytes

The liver is a metabolism and transfer center of amino acids as well as the prime target organ of insulin. In this report, we characterized the regulation of system N/A transporter 3 (SNAT3) in the liver of dietary-restricted mice and in hepatocytes treated with serum starvation and insulin. The expression of SNAT3 was up-regulated in dietary-restricted mice. The expression of SNAT3 protein was detected on the plasma membrane of hepatocyte-like H2.35 cells with a half-life of 6-8 h. When H2.35 cells were depleted of serum, the expression of SNAT3 was increased. An increased concentration of insulin, however, suppressed SNAT3 expression. Interestingly, the down-regulation of SNAT3 expression by insulin was blocked by the specific phosphoinositide 3-kinase inhibitor LY294002 and mammalian target of rapamycin inhibitor, but not by MAPK inhibitor PD98059, suggesting that insulin exerts its effect on SNAT3 through phosphoinositide 3-kinase-mammalian target of rapamycin signaling. Surface biotinylation assay showed an increased level of SNAT3 on the cell surface after 0.5 h of insulin treatment, although no effect was observed after 24 h of treatment. Consistently, the transport of the substrate l-histidine was increased with short, but not long, treatment by insulin in both H2.35- and SNAT3-transfected COS-7 cells. The L-histidine uptake was inhibited significantly by L-histidine followed by 2-endoamino-bicycloheptane-2-carboxylic acid and L-cysteine and to a lesser extent by L-alanine and aminoisobutyric acid, but was not inhibited by alpha-(methylamino)isobutyric acid, implying that uptake of L-histidine in H2.35 cells is primarily mediated by system N transporters. In conclusion, differential regulation of SNAT3 by insulin and serum starvation reinforces the functional significance of this transporter in liver physiology.     

Unimpaired membrane dynamics in parathyroid cells in insulin deficient rats

The responsiveness of parathyroid cells in insulin deficient and short-term diabetic rats was investigated by morphometric analysis. Insulin deficiency was produced by intravenous injection of D-mannoheptulose and short-term (7 days) diabetes mellitus by intraperitoneal application of streptozotocin. Parathyroid glands were stimulated for parathyroid hormone secretion by decreasing the serum calcium concentration through intravenous infusion of EGTA. Parathyroid cells of controls, insulin deficient, and short-term diabetic rats responded to reduced serum calcium by a 45% increase of the cell surface area. This increase is assumed to be the result of the membrane-bound transport of parathyroid hormone from the Golgi complex and secretory granules to the plasma membrane and subsequent exocytic release of parathyroid hormone induced by the low serum calcium concentration. Therefore, the unimpaired increase in the cell surface area of parathyroid cells in insulin deficient and short-term diabetic rats indicates that insulin does not modulate the release of parathyroid hormone. It is also considered likely that synthesis of parathyroid hormone is not suppressed in short-term diabetes but that fat metabolism is disturbed leading to accumulation of lipid vacuoles.     

Serum insulin,glucose and lactate concentrations during 18-h fast in female reindeer

We investigated whether secretion of insulin occurred in the absence of feeding in a ruminant. Serum insulin, glucose and lactate concentrations were measured in three adult non-pregnant reindeer at hourly intervals during an 18-h fast (17:30-11:30 h) in October. Mean serum insulin concentration was 39+/-3 micro/ml (range 2-100). The insulin profile of two animals was characterized by a nocturnal rise and an early morning trough, followed by a mid-morning rise. Within the larger peaks, short-term oscillations occurred at 2-3-h intervals. Serum glucose concentrations significantly increased during the fast and exceeded feeding values by 31-45% at 18 h post feeding. Serum lactate concentrations declined significantly in all three animals. Residuals for serum glucose concentrations were significantly negatively correlated to residuals of serum lactate in two animals, but not the third. Serum glucose and lactate concentrations were not related to serum insulin. In conclusion, insulin secretion in reindeer shows a 2-3-h periodicity in the absence of feeding. The periodicity is of similar duration as the inter-meal interval for pen-fed reindeer during winter (2.5 h). Although not necessarily causal, the results are consistent with a hypothesized role for insulin in meal initiation.     

Glycogen synthesis in the perfused liver of adrenalectomized rats.

1. A total loss of capacity for net glycogen synthesis was observed in experiments with the perfused liver of starved adrenalectomized rats. 2. This lesion was corrected by insulin or cortisol in vivo (over 2-5h), but not by any agent tested in perfusion. 3. The activity of glycogen synthetase a, and its increase during perfusion, in the presence of glucose plus glucogenic substrates, were proportional to the rate of net glycogen accumulation. 4. This complete inherent loss of capacity for glycogen synthesis after adrenalectomy is greater than any defect in hepatic metabolism yet reported in this situation, and is not explicable by a decrease in the rate of gluconegenesis (which supports glycogen synthesis in the liver of starved rats). The short-term (2-5h) stimulatory effect of glucocorticoids in the intact animal, on hepatic glycogen deposition, may be mediated partly through insulin action, although neither insulin or cortisol appear to act directly on the liver to stimulate glycogen synthesis.     

Regulation of caspase-3 activity by insulin in skeletal muscle cells involves both PI3-kinase and MEK-1/2

A hallmark of skeletal muscle atrophy is increased activities of several proteolytic systems, including caspase-3. We have previously shown that conditions involving insulin deficiency or insulin resistance increase both overall protein degradation and caspase-3-mediated actin cleavage. In the present experiments, we examined how insulin regulates caspase-3 activity in L6 myotubes. Reducing the serum concentration in the culture media from 2 to 0.5% overnight increased caspase-3 activity and actin cleavage. Addition of insulin to proteolytically active cells attenuated both responses within 4 h. Individually, inhibitors of either phosphatidylinositide 3-kinase (PI3K) or MEK1/2 partially blocked the insulin-induced reduction in caspase-3 activity; in combination, the inhibitors completely prevented insulin from attenuating caspase-3 activity. Insulin suppressed caspase-3 activity by a complex mechanism that included direct inhibition due to an increased interaction between caspase-3 and cellular inhibitor of apoptosis-1 and indirect inhibition via phosphorylation (i.e., inactivation) of the proapoptotic protein Bad, which participates in the intrinsic (i.e., mitochondrial) apoptosis activation cascade. Unlike other cell types, the phosphorylation of Bad Ser112 was mediated by the PI3K/Akt pathway rather than the MEK/ERK/ribosomal S6 protein kinase pathway. In summary, our findings indicate that insulin regulates caspase-3 activity by a multistep process that is unique to skeletal muscle, thus providing insights about the muscle-specific nature of the atrophy process.     

The Impact of Low-Dose Insulin on Peripheral Nerve Insulin Receptor Signaling in Streptozotocin-Induced Diabetic Rats

Background

The precise mechanisms of the neuroprotective effects of insulin in streptozotocin (STZ)-induced diabetic animals remain unknown, but altered peripheral nerve insulin receptor signaling due to insulin deficiency might be one cause.

Methodology and Principal Findings

Diabetes was induced in 10-week-old, male Wistar rats by injecting them with STZ (45 mg/kg). They were assigned to one group that received half of an insulin implant (∼1 U/day; I-group, n = 11) or another that remained untreated (U-group, n = 10) for 6 weeks. The controls were age- and sex-matched, non-diabetic Wistar rats (C-group, n = 12). Low-dose insulin did not change haemoglobin A1c, which increased by 136% in the U-group compared with the C-group. Thermal hypoalgesia and mechanical hyperalgesia developed in the U-group, but not in the I-group. Sensory and motor nerve conduction velocities decreased in the U-group, whereas sensory nerve conduction velocity increased by 7% (p = 0.0351) in the I-group compared with the U-group. Western blots showed unaltered total insulin receptor (IR), but a 31% decrease and 3.1- and 4.0-fold increases in phosphorylated IR, p44, and p42 MAPK protein levels, respectively, in sciatic nerves from the U-group compared with the C-group. Phosphorylated p44/42 MAPK protein decreased to control levels in the I-group (p<0.0001).

Conclusions and Significance

Low-dose insulin deactivated p44/42 MAPK and ameliorated peripheral sensory nerve dysfunction in rats with STZ-induced diabetes. These findings support the notion that insulin deficiency per se introduces impaired insulin receptor signaling in type 1 diabetic neuropathy.     

Interleukin-1alpha inhibits insulin signaling with phosphorylating insulin receptor substrate-1 on serine residues in 3T3-L1 adipocytes

Proinflammatory cytokines are recently reported to inhibit insulin signaling causing insulin resistance. IL-1alpha is also one of the proinflammatory cytokines; however, it has not been clarified whether IL-1alpha may also cause insulin resistance. Here, we investigated the effects of IL-1alpha treatment on insulin signaling in 3T3-L1 adipocytes. IL-1alpha treatment up to 4 h did not alter insulin-stimulated insulin receptor tyrosine phosphorylation, whereas tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and the association with phosphatidylinositol 3-kinase were partially inhibited with the maximal inhibition in around 15 min. IRS-1 was transiently phosphorylated on some serine residues around 15 min after IL-1alpha stimulation, when several serine kinases, IkappaB kinase, c-Jun-N-terminal kinase, ERK, and p70S6K were activated. Chemical inhibitors for these kinases inhibited IL-1alpha-induced serine phosphorylation of IRS-1. Tyrosine phosphorylation of IRS-1 was recovered only by the IKK inhibitor or JNK inhibitor, suggesting specific involvement of these two kinases. Insulin-stimulated Akt phosphorylation and 2-deoxyglucose uptake were not inhibited only by IL-1alpha. Interestingly, Akt phosphorylation was synergistically inhibited by IL-1alpha in the presence of IL-6. Taken together, short-term IL-1alpha treatment transiently causes insulin resistance at IRS-1 level with its serine phosphorylation. IL-1alpha may suppress insulin signaling downstream of IRS-1 in the presence of other cytokines, such as IL-6.     

UCP-2 and UCP-3 proteins are differentially regulated in pancreatic beta-cells

Background

Increased uncoupling protein-2 (UCP-2) expression has been associated with impaired insulin secretion, whereas UCP-3 protein levels are decreased in the skeleton muscle of type-2 diabetic subjects. In the present studies we hypothesize an opposing effect of glucose on the regulation of UCP-2 and UCP-3 in pancreatic islets.

Methodology

Dominant negative UCP-2 and wild type UCP-3 adenoviruses were generated, and insulin release by transduced human islets was measured. UCP-2 and UCP-3 mRNA levels were determined using quantitative PCR. UCP-2 and UCP-3 protein expression was investigated in human islets cultured in the presence of different glucose concentrations. Human pancreatic sections were analyzed for subcellular localization of UCP-3 using immunohistochemistry.

Principal Findings

Dominant negative UCP-2 expression in human islets increased insulin secretion compared to control islets (p<0.05). UCP-3 mRNA is expressed in human islets, but the relative abundance of UCP-2 mRNA was 8.1-fold higher (p<0.05). Immunohistochemical analysis confirmed co-localization of UCP-3 protein with mitochondria in human beta-cells. UCP-2 protein expression in human islets was increased ∼2-fold after high glucose exposure, whereas UCP-3 protein expression was decreased by ∼40% (p<0.05). UCP-3 overexpression improved glucose-stimulated insulin secretion.

Conclusions

UCP-2 and UCP-3 may have distinct roles in regulating beta-cell function. Increased expression of UCP-2 and decreased expression of UCP-3 in humans with chronic hyperglycemia may contribute to impaired glucose-stimulated insulin secretion. These data imply that mechanisms that suppress UCP-2 or mechanisms that increase UCP-3 expression and/or function are potential therapeutic targets to offset defects of insulin secretion in humans with type-2 diabetes.     

Diet Is Critical for Prolonged Glycemic Control after Short-Term Insulin Treatment in High-Fat Diet-Induced Type 2 Diabetic Male Mice

Background

Clinical studies suggest that short-term insulin treatment in new-onset type 2 diabetes (T2DM) can promote prolonged glycemic control. The purpose of this study was to establish an animal model to examine such a “legacy” effect of early insulin therapy (EIT) in long-term glycemic control in new-onset T2DM. The objective of the study was to investigate the role of diet following onset of diabetes in the favorable outcomes of EIT.

Methodology

As such, C57BL6/J male mice were fed a high-fat diet (HFD) for 21 weeks to induce diabetes and then received 4 weeks of daily insulin glargine or sham subcutaneous injections. Subsequently, mice were either kept on the HFD or switched to a low-fat diet (LFD) for 4 additional weeks.

Principal Findings

Mice fed a HFD gained significant fat mass and displayed increased leptin levels, increasing insulin resistance (poor HOMA-IR) and worse glucose tolerance test (GTT) performance in comparison to mice fed a LFD, as expected. Insulin-treated diabetic mice but maintained on the HFD demonstrated even greater weight gain and insulin resistance compared to sham-treated mice. However, insulin-treated mice switched to the LFD exhibited a better HOMA-IR compared to those mice left on a HFD. Further, between the insulin-treated and sham control mice, in spite of similar HOMA-IR values, the insulin-treated mice switched to a LFD following insulin therapy did demonstrate significantly better HOMA-B% values than sham control and insulin-treated HFD mice.

Conclusion/Interpretation

Early insulin treatment in HFD-induced T2DM in C57BL6/J mice was only beneficial in animals that were switched to a LFD after insulin treatment which may explain why a similar legacy effect in humans is achieved clinically in only a portion of cases studied, emphasizing a vital role for diet adherence in diabetes control.     

Regulation of carnitine palmitoyltransferase by insulin results in decreased activity and decreased apparent Ki values for malonyl-CoA

Administration to normal rats of 100 mg of streptozotocin/kg body weight produced ketotic diabetic rats in which the affinity of carnitine palmitoyltransferase for malonyl-CoA was decreased by 10-fold and its activity was increased by 30%, but the injection of insulin brought the affinity and the activity back to normal within 4 h. Administration of 60 mg of streptozotocin/kg produced non-ketotic diabetic rats and caused a less substantial change in the affinity of carnitine palmitoyltransferase for malonyl-CoA. In the BB Wistar diabetic rat, the onset of diabetes also increased the activity of carnitine palmitoyltransferase and decreased its affinity for malonyl-CoA. Injection of insulin brought both of these values back to normal within 2 h. The total activity of mitochondrial carnitine palmitoyltransferase (outer + inner activities) was 40% greater in the BB Wistar diabetic rat, but treatment with insulin did not decrease the total activity to normal values within 2 h. The elevated activity and decreased affinity for malonyl-CoA found in fasting rats did not respond to short-term insulin treatment. The evaluation of a previous report that cycloheximide blocks the effects of starvation indicated that cycloheximide did not act by inhibiting protein synthesis, but produced its effect by preventing gastric emptying. Current data suggest that diabetes increases the activity of carnitine palmitoyltransferase and greatly diminishes the affinity of the enzyme for malonyl-CoA and that the severity of diabetes is associated with differences in the affinity of the enzyme for its inhibitor. Insulin acts on the outer carnitine palmitoyltransferase to reverse these effects very rapidly, but diabetes produces some change in the total activity that is not reversed by short-term treatment with insulin.