Hypoglycemic episodes exacerbate skeletal muscle insulin resistance in type 2 diabetes mellitus

Abstract

To date, the specific impact of hypoglycemia on insulin resistance in skeletal muscle is unclear. This study aimed to investigate the correlation between hypoglycemia in individuals with Type 2 Diabetes Mellitus by assessing peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and Akt levels in both skeletal muscle tissue and blood plasma. A true experimental-design study was conducted at the Laboratory of Animal Clinical Study, Faculty of Veterinary, Universitas Syiah Kuala, from October to December 2022. The study utilized 24 male rats (Rattus norvegicus) and the rates were categorized into the following groups: control (K1), streptozocin-induced diabetic without hypoglycemia (K2), streptozocin-induced diabetic with mild hypoglycemia (K3), and streptozocin-induced diabetic with severe hypoglycemia (K4). The rats were euthanized a minimum of 30 min after hypoglycemia was confirmed. W Quadriceps femoris muscle and 2 ml of heart blood were collected. PGC-1α and protein kinase B/Akt were examined using enzyme-linked immunosorbent assay. This study demonstrates that PGC-1α and Akt levels in skeletal muscle and plasma are influenced by hypoglycemia severity in rats, with lower levels associated with more severe hypoglycemia, though no significant differences were observed between mild and severe hypoglycemia groups. A positive correlation was found between PGC-1α and Akt levels in skeletal muscle and plasma, suggesting interdependency. Control group plasma levels were 2.48 ± 0.53 ng/ml for PGC-1α and 11.26 ± 1.21 ng/ml for Akt. Even mild episodes of hypoglycemia can lead to a substantial deterioration of insulin resistance in skeletal muscle.

INTRODUCTION

Diabetes is a global health concern due to its high mortality and morbidity – approximately over half a billion people living with diabetes worldwide.[1,2] In 2021, type 2 diabetes mellitus (T2DM) accounted for 90% of all diabetes prevalence.[3] Thus, there is an urgent need to identify the mechanisms of insulin resistance and effective interventions for treating these metabolic diseases. The pathogenesis of T2DM often starts with insulin resistance, prompting increased β-cell insulin secretion to maintain normal blood glucose levels.[4] Over time, β-cell function and mass progressively decline, leading to insufficient insulin secretion and exacerbates the inability to compensate for insulin resistance, ultimately resulting in overt diabetes.[4]

Hyperglycemia causes glucotoxicity in the liver and worsens insulin resistance;[5] however, to the best of our knowledge, no research has been investigated regarding the effect of hypoglycemia on insulin resistance in skeletal muscle. Hypoglycemia is a common complication in diabetic patients, especially those undergoing treatment with insulin, sulfonylurea, or glinide medications, with reported rates of severe hypoglycemia averaging around 2.5 events per person per year.[6,7] Skeletal muscle plays a critical role in glucose clearance, accounting for more than 80% of glucose uptake following postprandial glucose elevation.[8] Consequently, when skeletal muscle exhibits good insulin sensitivity, it implies efficient glucose uptake by the skeletal muscle. Conversely, if insulin resistance in skeletal muscle worsens, it becomes challenging to mitigate postprandial hypoglycemia.

In insulin-dependent glucose uptake, a pathway involving the translocation of Glucose Transporter 4 (GLUT4) to skeletal muscle cell surfaces is critical.[9] Protein kinase B/Akt plays a central role.[8] Insulin resistance reduces tyrosine phosphorylation of insulin receptor substrates (IRS) 1 and IRS 2 while increasing serine/threonine phosphorylation.[10] Furthermore, proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) is a key regulator in skeletal muscle function, promoting mitochondrial and blood vessel growth, enhancing oxygen consumption, and fatigue resistance during skeletal muscle contractions.[11] Mitochondrial responses to metabolic changes affect cellular energy processes, impacting mitochondrial growth and contributing to insulin resistance and mitophagy.[12] In T2DM, PGC-1α regulation, especially in oxidative phosphorylation, is diminished.[8]

To date, the specific impact of hypoglycemia on insulin resistance in skeletal muscle is unclear, whether it is driven by insulin-related mechanisms involving the GLUT 4 response to Akt, or noninsulin-related factors, particularly a reduction in mitochondrial gene expression. This study aimed to investigate the correlation between hypoglycemia in individuals with T2DM by assessing PGC-1α and Akt levels in both skeletal muscle tissue and blood plasma.

METHODS

Study design and animal preparation

A true experimental-design study was conducted at the Laboratory of Animal Clinical Study, Faculty of Veterinary, Universitas Syiah Kuala, from October to December 2022. The study utilized 24 male rats (Rattus norvegicus) weighing 160–220 g and aged 8–12 weeks. Rats were included based on the following criteria: good motility, healthy white fur, clear eyes, and no existing physical impairments.

After a one-week acclimatization period, the rates were randomized with a web-based randomization process to allocate into the following groups: control (K1), streptozocin-induced diabetic without hypoglycemia (K2), streptozocin-induced diabetic with mild hypoglycemia (K3), and streptozocin-induced diabetic with severe hypoglycemia (K4). The veterinary and laboratory assistants involved in this study were blinded by using the same-look cage for each groups. Fasting blood glucose levels were assessed before treatment to determine that rats did not already have diabetes mellitus. Rats were fasted for 12 h and only allowed to drink, before the initial blood glucose measurement to stabilize their glycemic levels. Blood glucose was measured using a glucometer. A blood sample of 2.5–4 µL was applied to the test strip and results were displayed on the glucometer after 11 s. A detail procedure of the study is presented in Figure 1.

Figure 1.

Study workflow. PGC-1α: Peroxisome proliferator-activated receptor γ co-activator 1 α; ELISA: Enzyme-linked immunosorbent assay

Diabetic-induced procedure

In groups K2–K4, rats were induced into type 2 diabetes-like state by providing a high-fat diet for 2 weeks, followed by a 30 mg/kg intraperitoneal streptozocin injection.[13] Rats had unrestricted access to water and were fed twice daily, totaling up to 10%–15% of their body weight. Blood glucose levels were examined 5 days postinjection; levels exceeding 200 mg/dl indicated T2DM.

Hypoglycemia-induced procedure and tissue harvesting

Aspart Insulin was intraperitoneally administered to induce mild and severe hypoglycemia in K3 and K4 groups, respectively. Blood glucose levels were monitored every 15 min. Mild hypoglycemia is defined as a blood glucose level of <70 mg/dL, whereas severe hypoglycemia is defined as a blood glucose level of <50 mg/dL, following insulin administration. The rats were euthanized no sooner than 30 min following confirmation of hypoglycemia. The euthanasia procedure was carried out through cervical dislocation, performed by a veterinarian. The rats were then surgically removed from the femoral part. The incision progressed through the skin and subcutaneous tissue. Quadriceps femoris muscle was then isolated, minced, and homogenized in a 1:9 ratio with phosphate buffer saline. Further disruption of cells was achieved through sonication method.[14] After centrifugation (5 min at 5000 rpm), the skeletal muscle tissue samples (supernatant) were collected and stored at 4°C or −20°C. Moreover, 2 ml of heart blood was also collected for blood plasma measurement of PGC-1α and protein kinase B/Akt.

Proliferator-activated receptor gamma coactivator 1-alpha dan Akt enzyme measurement

PGC-1α and protein kinase B/Akt were examined using enzyme-linked immunosorbent assay (ELISA) kits (BioEnzy, Jakarta, Indonesia). Rats PGC-1α and protein kinase B antibodies were pre-coated on ELISA plates, followed by sample addition (plasma or muscle supernatant). Biotinylated Rat PGC-1α or protein kinase B/Akt antibodies and Streptavidin-HRP were added and incubated at 37°C for 60 min. After washing, substrate solution was added, color developed based on PGC-1α or protein kinase B/Akt levels, and absorbance was measured at 450 nm using an ELISA reader.[15]

Statistical analysis

The univariate analysis presented the frequency distribution of the baseline characteristics.

Four-group differences in PGC-1α and Akt values were analyzed with one-way ANOVA. Two-group differences were assessed with the Mann–Whitney test. Pearson’s test was used to analyze the correlation between PGC-1α in skeletal muscle/blood plasma and Akt levels in skeletal muscle/blood plasma. All statistical analyses were conducted using the SPSS software, version 25.0. (IBM SPSS, Chicago, IL, USA).

Ethical clearance

This study has obtained ethical clearance from the Institutional of Animal Care and Use Committee, School of Veterinary, Universitas Syiah Kuala with registered number 187/KEPH/XI/2022.

RESULTS

Table 1 shows no significant differences in initial and postacclimatization body weights among treatment groups. All rats had fasting glucose levels below 140 mg/dl before the intervention, indicating no preexisting diabetes. The severe hypoglycemia group (K4) took significantly longer to reach the target hypoglycemia compared to the mild hypoglycemia group (K3). No adverse events were observed during the study.

Table 1.

Baseline characteristic of rats among various treatment groups in the present study (n=28)

Variables		Treatment group, median (minimum–maximum)								P
		K1		K2		K3		K4
Initial weight (g)		196 (178–216)		192 (164–207)		193 (178–205)		189 (161–191)		0.14a
Body weight after acclimatization (g)		234 (198–257)		223 (198–238)		202 (195–225)		219 (200–239)		0.09a
Fasting blood glucose levels before intervention (mg/dL)		136 (128–138)		125 (99–131)		124 (112–130)		128 (118–133)		0.06a
Blood glucose levels after 5 days of streptozocin administration (mg/dL)		132 (123–143)		Hi* (250–Hi*)		698 (233–Hi*)		472 (316–Hi*)		0.00a
Time to achieve hypoglycemia target (min)						345 (330–390)		720 (510–780)		0.00b

*Glucometer result showed glucose level >500 mg/dL, ^aOne-way ANOVA test, ^bMann–Whitney test. K1: Control group, K2: Streptocozin-induced diabetic and without hypoglycemia, K3: Streptocozin-induced diabetic and mild hypoglycemia, K4: Streptocozin-induced diabetic and severe hypoglycemia

Differences in skeletal muscle and blood plasma proliferator-activated receptor gamma coactivator 1-alpha and Akt levels

The mean PGC-1α levels in skeletal muscle (ng/g) in groups K1, K2, K3, and K4 were 32.25 ± 4.18, 28.34 ± 3.85, 26.96 ± 3.31, and 22.97 ± 3.22, respectively, whereas plasma levels (ng/ml) were 2.48 ± 0.53, 2.16 ± 0.31, 1.76 ± 0.25, and 1.44 ± 0.19, respectively. Furthermore, the mean levels of skeleteal muscle Akt (ng/g) in groups K1, K2, K3, and K4 were 158.52 ± 21.67, 137.58 ± 12.64, 134.97 ± 12.94, and 130.09 ± 9.51, respectively, whereas the mean levels of plasma Akt (ng/ml) were 11.26 ± 1.21, 11.13 ± 1.31, 10.58 ± 1.10, and 8.61 ± 1.42, respectively. As shown in Figure 2, statistically significant differences (P < 0.05) were observed in PGC-1α and Akt levels among various treatment groups both in skeletal muscle and blood plasma. The data suggest that PGC-1α and Akt enzyme levels in both skeletal muscle tissue and blood plasma are influenced by the severity of hypoglycemia in rats, with more severe hypoglycemia associated with lower PGC-1α and Akt levels; however, no statistical differences were found between groups having mild and severe hypoglycemia.

Figure 2.

Dot plot graph of differences in proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and Akt levels in various treatment groups in muscle tissue and plasma. Statistically significant differences (P < 0.05) were observed in PGC-1α and Akt levels among various treatment groups both in skeletal muscle and blood plasma. K1: Control, K2: strePtozocin-induced diabetic without hypoglycemia, K3: Streptozocin-induced diabetic with mild hypoglycemia, K4: strePtozocin-induced diabetic with severe hypoglycemia

Correlation of proliferator-activated receptor gamma coactivator 1-alpha and Akt levels in skeletal muscle tissue and blood plasma

Figure 3 demonstrates strong positive correlations (r = 0.529, P = 0.008 for PGC-1α; r = 0.580, P = 0.004 for Akt) between PGC-1α and Akt levels in skeletal muscle tissue and blood plasma. These findings suggest that as PGC-1α and Akt levels increase in skeletal muscle tissue, a corresponding increase occurs in blood plasma, conversely, indicating an interdependency between PGC-1α and Akt levels in these two compartments.

Figure 3.

Dot plot correlation graph of proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and Akt levels in muscle tissue and plasma. Strong positive correlations were observed between PGC-1α and Akt levels in skeletal muscle tissue and blood plasma

DISCUSSION

This study aimed to investigate the involvement of PGC-1α and Akt in the pathogenesis of T2DM experiencing hypoglycemia episodes. PGC-1α functions as a transcriptional coactivator that is critically involved in the regulation of cellular energy metabolism.[16] It functions as a coactivator for peroxisome proliferator-activated receptor, estrogen related receptor, and nuclear respiratory factor 1, which are instrumental in promoting the biogenesis of new mitochondria by regulating genes responsible for mitochondrial oxidative phosphorylation.[16] In addition, PGC-1α aids in transforming skeletal muscle tissue into a more metabolically oxidative fiber type composition while reducing glycolytic metabolism.[17] Furthermore, it plays a part in controlling carbohydrate and lipid metabolism.[17] When PGC-1α levels decline, it leads to an increase in glycolysis capacity, a reduction in oxidative metabolism, and a decrease in the quantity of mitochondria.[18]

PGC-1α not only facilitates glucose uptake but also enhances efficient glucose utilization.[19] Hypoglycemia can worsen existing intracellular insulin signaling problems in individuals with type 2 diabetes. The present study revealed that the severity of hypoglycemia was associated with lower levels of PGC-1α and Akt enzymes in both skeletal muscle tissue and blood plasma. When PGC-1α and Akt levels increase in skeletal muscle tissue, a corresponding increase is observed in blood plasma. However, no statistical difference was found between mild and severe hypoglycemia. It may stem from several factors. First, the study’s sample size (six rats per group) might have been underpowered to detect subtle biological differences between these two conditions, particularly if the molecular responses to hypoglycemia reach a plateau beyond a certain threshold. Second, hypoglycemia-induced metabolic stress, even at mild levels, could trigger maximal compensatory downregulation of insulin signaling pathways, leaving little room for further reduction in severe cases. This suggests a potential biological “floor effect,” where PGC-1α and Akt levels are already suppressed to a critical minimum in mild hypoglycemia, and severe hypoglycemia does not exacerbate this further within the experimental timeframe. In addition, the 30-min posthypoglycemia observation period might have been insufficient to capture delayed or progressive changes in these markers, particularly in severe hypoglycemia, which could involve more complex recovery mechanisms. Methodological limitations, such as the sensitivity of ELISA assays or variability in insulin administration protocols, might also contribute to the lack of observed differences. Future studies with larger cohorts, extended observation periods, and advanced molecular profiling could clarify these dynamics and refine our understanding of hypoglycemia’s dose-dependent effects on skeletal muscle insulin resistance. However, this underscores the importance of preventing hypoglycemic episodes, as even mild instances can impact the metabolic activity of glucose in skeletal muscle. Furthermore, the present study findings indicate that hypoglycemic conditions have an impact on insulin signaling abnormalities.

Skeletal muscles play a substantial role in disposing of postprandial glucose uptake, particularly when stimulated by insulin.[20] The intracellular signaling mechanism responsible for glucose metabolism begins with the binding of insulin to its receptor, subsequently activating the IRS 1/phosphoinositide 3-kinase signaling pathway.[21] This activation subsequently activates Akt, inducing the phosphorylation of AS160, which facilitates the translocation of GLUT4 to the cell membrane, thereby enabling glucose uptake into muscle cells.[22] Once inside, glucose is metabolized through oxidative or nonoxidative pathways.[23]

This research highlights the importance of two key markers in the insulin signaling pathway affecting glucose regulation in skeletal muscle: Protein Kinase B/Akt and PGC-1α. However, a study by Jaiswal et al. found that reducing Akt activity solely in skeletal muscle is insufficient to induce insulin resistance – other signaling molecules beyond Akt in skeletal muscle are needed for insulin resistance.[24] Insulin resistance under normal conditions involves various factors, including insulin, insulin receptors, IRS, GLUT4, Akt, mitogen-activated protein kinase, and AMP-activated protein kinase, all intricately involved in regulating glucose and lipid metabolism in the body.[25]

CONCLUSION

Hypoglycemia, even mild hypoglycemia episodes, can cause a significant worsening of insulin resistance in skeletal muscle. These findings suggest that as PGC-1α and Akt levels increase in skeletal muscle tissue, a corresponding increase occurs in blood plasma, conversely, indicating an interdependency and shared regulatory mechanisms between PGC-1α and Akt. Understanding this correlation may offer insights into targeted treatments for insulin resistance and related complications in T2DM and the potential use of these biomarkers for diagnosis, though further research is needed to uncover the underlying mechanisms and potential interventions.

The findings of this study hold significant therapeutic implications for the management of T2DM and its associated complications. The observed reduction in PGC-1α and Akt levels in skeletal muscle and blood plasma during hypoglycemic episodes underscores the detrimental impact of hypoglycemia on insulin signaling and glucose metabolism. These results suggest that even mild hypoglycemia can exacerbate insulin resistance in skeletal muscle, highlighting the importance of preventing hypoglycemic episodes in diabetic patients, particularly those on insulin or sulfonylurea therapy. Therapeutic strategies aimed at stabilizing blood glucose levels and minimizing hypoglycemic events could help preserve skeletal muscle insulin sensitivity and improve overall metabolic health. Additionally, the strong positive correlation between PGC-1α and Akt levels in skeletal muscle and plasma indicates their interdependency and shared regulatory mechanisms, positioning them as potential biomarkers for assessing insulin resistance and metabolic dysfunction. Targeting these pathways through pharmacological or lifestyle interventions, such as enhancing mitochondrial function or modulating PGC-1α activity, may offer novel approaches to mitigating insulin resistance and improving glucose uptake in skeletal muscle. Further research is warranted to explore the underlying mechanisms and develop targeted therapies that leverage these findings to improve outcomes for individuals with T2DM.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.