Muscle wasting in insulinopenic rats results from activation of the ATP-dependent, ubiquitin-proteasome proteolytic pathway by a mechanism including gene transcription.

In normal subjects and diabetic patients, insulin suppresses whole body proteolysis suggesting that the loss of lean body mass and muscle wasting in insulinopenia is related to increased muscle protein degradation. To document how insulinopenia affects organ weights and to identify the pathway for accelerated proteolysis in muscle, streptozotocin-treated and vehicle-injected, pair-fed control rats were studied. The weights of liver, adipose tissue, and muscle were decreased while muscle protein degradation was increased 75% by insulinopenia. This proteolytic response was not eliminated by blocking lysosomal function and calcium-dependent proteases at 7 or 3 d after streptozotocin. When ATP synthesis in muscle was inhibited, the rates of proteolysis were reduced to the same level in insulinopenic and control rats suggesting that the ATP-dependent, ubiquitin-proteasome pathway is activated. Additional evidence for activation of this pathway in muscle includes: (a) an inhibitor of proteasome activity eliminated the increased protein degradation; (b) mRNAs encoding ubiquitin and proteasome subunits were increased two- to threefold; and (c) there was increased transcription of the ubiquitin gene. We conclude that the mechanism for muscle protein wasting in insulinopenia includes activation of the ubiquitin-proteasome pathway with increased expression of the ubiquitin gene.

Protein degradation and increased mRNAs encoding proteins of the ubiquitin-proteasome proteolytic pathway in BC3H1 myocytes require an interaction between glucocorticoids and acidification.

In rats and humans, metabolic acidosis stimulates protein degradation and glucocorticoids have been implicated in this response. To evaluate the importance of glucocorticoids in stimulating proteolysis, we measured protein degradation in BC3H1 myocytes cultured in 12% serum. Acidification accelerated protein degradation but dexamethasone did not augment this response. To reduce the influence of glucocorticoids and other hormones and cytokines in 12% serum that could mediate proteolysis, we studied BC3H1 myocytes maintained in only 1% serum. Acidification of the medium or addition of dexamethasone at pH 7.4 did not significantly increase protein degradation, while acidification plus dexamethasone accelerated proteolysis. The steroid receptor antagonist RU 486 prevented this proteolytic response. Acidification of the medium with 1% serum did increase the mRNAs for ubiquitin and the C2 proteasome subunit, but when dexamethasone was added the mRNAs were increased significantly more. The steroid-receptor antagonist RU 486 suppressed this response to the addition of dexamethasone but the mRNAs remained at the levels measured in cells at pH 7.1 alone. Thus, acidification alone can increase the mRNAs of the ubiquitin-proteasome proteolytic pathway, but both acidosis and glucocorticoids are required to stimulate protein degradation. Since these changes occur without adding cytokines or other hormones, we conclude that the proteolytic response to acidification requires glucocorticoids.

The acidosis of chronic renal failure activates muscle proteolysis in rats by augmenting transcription of genes encoding proteins of the ATP-dependent ubiquitin-proteasome pathway.

Chronic renal failure (CRF) is associated with negative nitrogen balance and loss of lean body mass. To identify specific proteolytic pathways activated by CRF, protein degradation was measured in incubated epitrochlearis muscles from CRF and sham-operated, pair-fed rats. CRF stimulated muscle proteolysis, and inhibition of lysosomal and calcium-activated proteases did not eliminate this increase. When ATP production was blocked, proteolysis in CRF muscles fell to the same level as that in control muscles. Increased proteolysis was also prevented by feeding CRF rats sodium bicarbonate, suggesting that activation depends on acidification. Evidence that the ATP-dependent ubiquitin-proteasome pathway is stimulated by the acidemia of CRF includes the following findings: (a) An inhibitor of the proteasome eliminated the increase in muscle proteolysis; and (b) there was an increase in mRNAs encoding ubiquitin (324%) and proteasome subunits C3 (137%) and C9 (251%) in muscle. This response involved gene activation since transcription of mRNAs for ubiquitin and the C3 subunit were selectively increased in muscle of CRF rats. We conclude that CRF stimulates muscle proteolysis by activating the ATP-ubiquitin-proteasome-dependent pathway. The mechanism depends on acidification and increased expression of genes encoding components of the system. These responses could contribute to the loss of muscle mass associated with CRF.

Necessary but not sufficient: the role of glucocorticoids in the acidosis-induced increase in levels of mRNAs encoding proteins of the ATP-dependent proteolytic pathway in rat muscle.

Muscle protein degradation is accelerated by the acidosis associated with chronic renal failure. In isolated muscles from acidotic rats, a cytosolic, ATP-dependent proteolytic pathway is stimulated with a concurrent increase in the abundance of mRNAs encoding ubiquitin and subunits of the 26S proteasome complex associated with this degradative pathway. Adrenalectomy (ADX) prevents the acidosis-induced increase in muscle protein degradation unless high physiologic doses of glucocorticoids are administered to acidotic, adrenalectomized rats. We have examined the roles that acidosis and glucocorticoids have in the increase in mRNAs encoding proteins of the ATP-dependent-ubiquitin-proteasome proteolytic pathway in ADX rats. We found that ubiquitin and proteasome C2 and C9 subunit mRNA levels are increased in the white fiber, extensor digitorus longus (EDL) and mixed fiber, gastrocnemius muscles from acidotic ADX rats that received dexamethasone whereas acidosis alone or dexamethasone alone failed to increase these mRNAs. In contrast, acidosis plus dexamethasone decreased the total RNA content in both muscles. These data suggest that in muscle, the response to acidosis involves the specific activation of the ATP-ubiquitin-proteasome proteolytic pathway. Moreover, glucocorticoids are required but not directly responsible for the acidosis-induced increase in the mRNAs encoding proteins of this degradative pathway.

Acidosis and glucocorticoids concomitantly increase ubiquitin and proteasome subunit mRNAs in rat muscle.

In rat muscle metabolic acidosis increases ATP-dependent protein degradation and levels of mRNAs for ubiquitin (Ub) and proteasome subunits. Because adrenalectomy (ADX) abolishes the proteolytic response to acidosis in muscle, we examined whether glucocorticoids (GCs) are necessary for acidosis-induced changes in Ub and proteasome mRNAs in muscles. Total RNA content of the white fiber extensor digitorum longus or mixed fiber gastrocnemius muscles were lowest in muscles of ADX rats given acid plus GCs. In contrast, the abundance of Ub and C2 and C9 proteasome subunits mRNAs were increased in muscles from this group compared with untreated ADX rats or ADX rats given acid or GCs alone. Because total RNA is reduced, the increase in these mRNAs in muscles of ADX rats receiving acid plus GCs provides evidence for a specific activation of the ATP-dependent-Ub-proteasome pathway. Thus, GCs are required but not sufficient to produce the coordinated increase in mRNAs encoding ubiquitin and proteasome subunits occurring in muscles of acidotic rats.

Metabolic acidosis stimulates muscle protein degradation by activating the adenosine triphosphate-dependent pathway involving ubiquitin and proteasomes.

Metabolic acidosis often leads to loss of body protein due mainly to accelerated protein breakdown in muscle. To identify which proteolytic pathway is activated, we measured protein degradation in incubated epitrochlearis muscles from acidotic (NH4Cl-treated) and pair-fed rats under conditions that block different proteolytic systems. Inhibiting lysosomal and calcium-activated proteases did not reduce the acidosis-induced increase in muscle proteolysis. However, when ATP production was also blocked, proteolysis fell to the same low level in muscles of acidotic and control rats. Acidosis, therefore, stimulates selectively an ATP-dependent, nonlysosomal, proteolytic process. We also examined whether the activated pathway involves ubiquitin and proteasomes (multicatalytic proteinases). Acidosis was associated with a 2.5- to 4-fold increase in ubiquitin mRNA in muscle. There was no increase in muscle heat shock protein 70 mRNA or in kidney ubiquitin mRNA, suggesting specificity of the response. Ubiquitin mRNA in muscle returned to control levels within 24 h after cessation of acidosis. mRNA for subunits of the proteasome (C2 and C3) in muscle were also increased 4-fold and 2.5-fold, respectively, with acidosis; mRNA for cathepsin B did not change. These results are consistent with, but do not prove that acidosis stimulates muscle proteolysis by activating the ATP-ubiquitin-proteasome-dependent, proteolytic pathway.

Na pump defects in chronic uremia cannot be attributed to changes in Na-K-ATPase mRNA or protein.

We have found abnormalities in Na-K-adenosine-triphosphatase (Na-K-ATPase) function in different tissues of rats with chronic renal failure (CRF). A potential mechanism for these findings is a change in Na-K-ATPase alpha- and/or beta-gene expression. To evaluate this possibility, we compared CRF with pair-fed, sham-operated rats to determine whether chronic uremia changes the expression of Na-K-ATPase alpha 1-, alpha 2-, beta 1-, and beta 2-isoform mRNAs or protein in different types of skeletal muscle, heart, liver, adipose, and kidney tissue. In CRF rats, alpha 1-mRNA in heart tended to be higher and beta 2-mRNA was lower in fat and kidney. There were no other statistically significant differences in isoform mRNAs in tissues of CRF compared with the control rats. Western blot analysis revealed a 38% increase in alpha 1-protein in adipocytes and a 61% decrease in kidney of CRF rats but no significant differences in the amounts of isoform protein in other tissues. Thus, in uremia, posttranslational events or inhibitors of the enzyme are more likely causes of defects in Na-K-ATPase than changes in mRNA or protein abundance.

Mechanisms that cause protein and amino acid catabolism in uremia.

Anorexia and/or a protein- and calorie-restricted diet can cause protein wasting by limiting the intake of essential amino acids (EAA) and, hence, protein synthesis. By this mechanism plus the effects of inadequate calories, restricted diets could contribute to the loss of lean body mass of uremic patients. Uremia also impairs the normal metabolic responses that must be activated to preserve body protein, thereby augmenting the adverse effects of anorexia. The responses impaired are those that conserve EAA and protein, which results in catabolism of EAA and muscle protein. An important factor that initiates abnormal adaptive responses in uremia is metabolic acidosis, because acidosis stimulates muscle protein degradation and increases the activity of branched-chain ketoacid dehydrogenase and, hence, the catabolism of branched-chain amino acids (BCAA). The effects of acidosis could be mediated by impaired regulation of intracellular pH and/or an increase in glucocorticoid production. Research directed at identifying the specific proteolytic pathways that are activated by metabolic acidosis has excluded a major role for Ca(2+)-activated or lysosomal proteases and suggests activation of an adenosine triphosphate (ATP)- and ubiquitin-dependent proteolytic pathway. The mechanism of activation of this pathway includes an increase in mRNA for enzymes involved in protein and amino acid catabolism.