Central Acting Hsp10 Regulates Mitochondrial Function, Fatty Acid Metabolism, and Insulin Sensitivity in the Hypothalamus.

Mitochondria are critical for hypothalamic function and regulators of metabolism. Hypothalamic mitochondrial dysfunction with decreased mitochondrial chaperone expression is present in type 2 diabetes (T2D). Recently, we demonstrated that a dysregulated mitochondrial stress response (MSR) with reduced chaperone expression in the hypothalamus is an early event in obesity development due to insufficient insulin signaling. Although insulin activates this response and improves metabolism, the metabolic impact of one of its members, the mitochondrial chaperone heat shock protein 10 (Hsp10), is unknown. Thus, we hypothesized that a reduction of Hsp10 in hypothalamic neurons will impair mitochondrial function and impact brain insulin action. Therefore, we investigated the role of chaperone Hsp10 by introducing a lentiviral-mediated Hsp10 knockdown (KD) in the hypothalamic cell line CLU-183 and in the arcuate nucleus (ARC) of C57BL/6N male mice. We analyzed mitochondrial function and insulin signaling utilizing qPCR, Western blot, XF96 Analyzer, immunohistochemistry, and microscopy techniques. We show that Hsp10 expression is reduced in T2D mice brains and regulated by leptin in vitro. Hsp10 KD in hypothalamic cells induced mitochondrial dysfunction with altered fatty acid metabolism and increased mitochondria-specific oxidative stress resulting in neuronal insulin resistance. Consequently, the reduction of Hsp10 in the ARC of C57BL/6N mice caused hypothalamic insulin resistance with acute liver insulin resistance.

Redox homeostasis and cell cycle activation mediate beta-cell mass expansion in aged, diabetes-prone mice under metabolic stress conditions: Role of thioredoxin-interacting protein (TXNIP).

Overnutrition contributes to insulin resistance, obesity and metabolic stress, initiating a loss of functional beta-cells and diabetes development. Whether these damaging effects are amplified in advanced age is barely investigated. Therefore, New Zealand Obese (NZO) mice, a well-established model for the investigation of human obesity-associated type 2 diabetes, were fed a metabolically challenging diet with a high-fat, carbohydrate restricted period followed by a carbohydrate intervention in young as well as advanced age. Interestingly, while young NZO mice developed massive hyperglycemia in response to carbohydrate feeding, leading to beta-cell dysfunction and cell death, aged counterparts compensated the increased insulin demand by persistent beta-cell function and beta-cell mass expansion. Beta-cell loss in young NZO islets was linked to increased expression of thioredoxin-interacting protein (TXNIP), presumably initiating an apoptosis-signaling cascade via caspase-3 activation. In contrast, islets of aged NZOs exhibited a sustained redox balance without changes in TXNIP expression, associated with higher proliferative potential by cell cycle activation. These findings support the relevance of a maintained proliferative potential and redox homeostasis for preserving islet functionality under metabolic stress, with the peculiarity that this adaptive response emerged with advanced age in diabetes-prone NZO mice.

Age-Related Maintenance of the Autophagy-Lysosomal System Is Dependent on Skeletal Muscle Type.

The skeletal muscle plays an important role in maintaining whole-body mechanics, metabolic homeostasis, and interorgan crosstalk. However, during aging, functional and structural changes such as fiber integrity loss and atrophy can occur across different species. A commonly observed hallmark of aged skeletal muscle is the accumulation of oxidatively modified proteins and protein aggregates which point to an imbalance in proteostasis systems such as degradation machineries. Recently, we showed that the ubiquitin-proteasomal system was impaired. Specifically, the proteasomal activity, which was declining in aged M. soleus (SOL) and M. extensor digitorum longus (EDL). Therefore, in order to understand whether another proteolytic system would compensate the decline in proteasomal activity, we aimed to investigate age-related changes in the autophagy-lysosomal system (ALS) in SOL, mostly consisting of slow-twitch fibers, and EDL, mainly composed of fast-twitch fibers, from young (4 months) and old (25 months) C57BL/6JRj mice. Here, we focused on changes in the content of modified proteins and the ALS. Our results show that aged SOL and EDL display high levels of protein modifications, particularly in old SOL. While autophagy machinery appears to be functional, lysosomal activity declines gradually in aged SOL. In contrast, in old EDL, the ALS seems to be affected, demonstrated by an increased level of key autophagy-related proteins, which are known to accumulate when their delivery or degradation is impaired. In fact, lysosomal activity was significantly decreased in old EDL. Results presented herein suggest that the ALS can compensate the high levels of modified proteins in the more oxidative muscle, SOL, while EDL seems to be more prone to ALS age-related alterations.

GPx3 dysregulation impacts adipose tissue insulin receptor expression and sensitivity.

Insulin receptor signaling is crucial for white adipose tissue (WAT) function. Consequently, lack of insulin receptor (IR) in WAT results in a diabetes-like phenotype. Yet, causes for IR downregulation in WAT of patients with diabetes are not well understood. By using multiple mouse models of obesity and insulin resistance, we identify a common downregulation of IR with a reduction of mRNA expression of selenoproteins Txnrd3, Sephs2, and Gpx3 in gonadal adipose tissue. Consistently, GPX3 is also decreased in adipose tissue of insulin-resistant and obese patients. Inducing Gpx3 expression via selenite treatment enhances IR expression via activation of the transcription factor Sp1 in 3T3-L1 preadipocytes and improves adipocyte differentiation and function. Feeding mice a selenium-enriched high-fat diet alleviates diet-induced insulin resistance with increased insulin sensitivity, decreased tissue inflammation, and elevated IR expression in WAT. Again, IR expression correlated positively with Gpx3 expression, a phenotype that is also conserved in humans. Consequently, decreasing GPx3 using siRNA technique reduced IR expression and insulin sensitivity in 3T3-L1 preadipocytes. Overall, our data identify GPx3 as a potentially novel regulator of IR expression and insulin sensitivity in adipose tissue.

Interplay of Dietary Fatty Acids and Cholesterol Impacts Brain Mitochondria and Insulin Action.

Overconsumption of high-fat and cholesterol-containing diets is detrimental for metabolism and mitochondrial function, causes inflammatory responses and impairs insulin action in peripheral tissues. Dietary fatty acids can enter the brain to mediate the nutritional status, but also to influence neuronal homeostasis. Yet, it is unclear whether cholesterol-containing high-fat diets (HFDs) with different combinations of fatty acids exert metabolic stress and impact mitochondrial function in the brain. To investigate whether cholesterol in combination with different fatty acids impacts neuronal metabolism and mitochondrial function, C57BL/6J mice received different cholesterol-containing diets with either high concentrations of long-chain saturated fatty acids or soybean oil-derived poly-unsaturated fatty acids. In addition, CLU183 neurons were stimulated with combinations of palmitate, linoleic acid and cholesterol to assess their effects on metabolic stress, mitochondrial function and insulin action. The dietary interventions resulted in a molecular signature of metabolic stress in the hypothalamus with decreased expression of occludin and subunits of mitochondrial electron chain complexes, elevated protein carbonylation, as well as c-Jun N-terminal kinase (JNK) activation. Palmitate caused mitochondrial dysfunction, oxidative stress, insulin and insulin-like growth factor-1 (IGF-1) resistance, while cholesterol and linoleic acid did not cause functional alterations. Finally, we defined insulin receptor as a novel negative regulator of metabolically stress-induced JNK activation.

Insulin action in the brain regulates mitochondrial stress responses and reduces diet-induced weight gain.

OBJECTIVE: Insulin action in the brain controls metabolism and brain function, which is linked to proper mitochondrial function. Conversely, brain insulin resistance associates with mitochondrial stress and metabolic and neurodegenerative diseases. In the present study, we aimed to decipher the impact of hypothalamic insulin action on mitochondrial stress responses, function and metabolism. METHODS: To investigate the crosstalk of insulin action and mitochondrial stress responses (MSR), namely the mitochondrial unfolded protein response (UPRmt) and integrated stress response (ISR), qPCR, western blotting, and mitochondrial activity assays were performed. These methods were used to analyze the hypothalamic cell line CLU183 treated with insulin in the presence or absence of the insulin receptor as well as in mice fed a high fat diet (HFD) for three days and STZ-treated mice without or with insulin therapy. Intranasal insulin treatment was used to investigate the effect of acute brain insulin action on metabolism and mitochondrial stress responses. RESULTS: Acute HFD feeding reduces hypothalamic mitochondrial stress responsive gene expression of Atf4, Chop, Hsp60, Hsp10, ClpP, and Lonp1 in C57BL/6N mice. We show that insulin via ERK activation increases the expression of MSR genes in vitro as well as in the hypothalamus of streptozotocin-treated mice. This regulation propagates mitochondrial function by controlling mitochondrial proteostasis and prevents excessive autophagy under serum deprivation. Finally, short-term intranasal insulin treatment activates MSR gene expression in the hypothalamus of HFD-fed C57BL/6N mice and reduces food intake and body weight development. CONCLUSIONS: We define hypothalamic insulin action as a novel master regulator of MSR, ensuring proper mitochondrial function by controlling mitochondrial proteostasis and regulating metabolism.